key: cord-0029162-tpeu7505
authors: Seo, Mi Hyun; Kim, Dae Won; Kim, Yeon Sook; Lee, Suk Keun
title: Pentoxifylline-induced protein expression change in RAW 264.7 cells as determined by immunoprecipitation-based high performance liquid chromatography
date: 2022-03-25
journal: PLoS One
DOI: 10.1371/journal.pone.0261797
sha: 0e637a00ebfa76280a09c2f2bd8a12122db5cc4c
doc_id: 29162
cord_uid: tpeu7505

Although pentoxifylline (PTX) was identified as a competitive non-selective phosphodiesterase inhibitor, its pharmacological effect has not been clearly elucidated. The present study explored the effect of low dose 10 μg/mL PTX (therapeutic dose) compared to high dose 300 μg/mL PTX (experimental dose) in RAW 264.7 cells through immunoprecipitation-based high performance liquid chromatography (IP-HPLC), immunohistochemistry, and western blot. 10 μg/mL PTX increased the expression of proliferation (Ki-67, PCNA, cyclin D2, cdc25A), epigenetic modification (KDM4D, PCAF, HMGB1), protein translation (DOHH, DHPS, eIF5A1), RAS signaling (KRAS, pAKT1/2/3, PI3K), NFkB signaling (NFkB, GADD45, p38), protection (HSP70, SOD1, GSTO1/2), survival (pAKT1/2/3, SP1, sirtuin 6), neuromuscular differentiation (NSEγ, myosin-1a, desmin), osteoblastic differentiation (BMP2, RUNX2, osterix), acute inflammation (TNFα, IL-1, CXCR4), innate immunity (β-defensin 1, lactoferrin, TLR-3, -4), cell-mediated immunity (CD4, CD8, CD80), while decreased the expression of ER stress (eIF2α, eIF2AK3, ATF6α), fibrosis (FGF2, CTGF, collagen 3A1), and chronic inflammation (CD68, MMP-2, -3, COX2) versus the untreated controls. The activation of proliferation by 10 μg/mL PTX was also supported by the increase of cMyc-MAX heterodimer and β-catenin-TCF1 complex in double IP-HPLC. 10 μg/mL PTX enhanced FAS-mediated apoptosis but diminished p53-mediated apoptosis, and downregulated many angiogenesis proteins (angiogenin, VEGF-A, and FLT4), but upregulated HIF1α, VEGFR2, and CMG2 reactively. Whereas, 300 μg/mL PTX consistently decreased proliferation, epigenetic modification, RAS and NFkB signaling, neuromuscular and osteoblastic differentiation, but increased apoptosis, ER stress, and fibrosis compared to 10 μg/mL PTX. These data suggest PTX has different biological effect on RWA 264.7 cells depending on the concentration of 10 μg/mL and 300 μg/mL PTX. The low dose 10 μg/mL PTX enhanced RAS/NFkB signaling, proliferation, differentiation, and inflammation, particularly, it stimulated neuromuscular and osteoblastic differentiation, innate immunity, and cell-mediated immunity, but attenuated ER stress, fibrosis, angiogenesis, and chronic inflammation, while the high dose 300 μg/mL PTX was found to alleviate the 10 μg/mL PTX-induced biological effects, resulted in the suppression of RAS/NFkB signaling, proliferation, neuromuscular and osteoblastic differentiation, and inflammation.

Introduction Pentoxifylline (PTX), a xanthine derivative, is primarily used as an antiproteolytic agent to treat muscle pain in people with peripheral artery disease [1, 2] by activating cAMP/EPAC/ AKT signaling [3, 4] . It has been frequently reported that PTX remarkably suppressed the secretions of pro-inflammatory cytokines and the nuclear factor-kappa B (NFkB) activation [5] [6] [7] , and reduced chronic inflammation [5, 8] . PTX appeared to have anti-fibrotic effect on radiation-induced lung fibrosis by modulation of PKA and PAI-1 expression as possible antifibrotic mechanisms [9] , and PTX therapy with vitamin E showed prevention of radiationinduced fibrosis in breast cancer patients [10] . PTX is suggested as an oral osteogenic drug for the treatment of post-menopausal osteoporosis [11] . As PTX given before tooth extraction is prophylactic, it might affect healing in a positive way by optimizing the inflammatory response [12] . Many authors suggest that PTX may increase the anticancer potential of anticancer drugs such as cisplatin or doxorubicin as well as reduce side effects of these drugs [13] [14] [15] [16] .

As RAW 264.7 cells are immortalized macrophages which are mainly involved with wound healing and tumor progression, the present study utilized RAW 264.7 cells for in vitro protein expression experiment. Although PTX has short half-life (0.39-0.84 h for the various doses and 0.96-1.61 h for the metabolites), its therapeutic dose for adult human is usually 400 mg (Trental), three times a day [17, 18] . Therefore, in this study, RAW 264.7 cells were primarily treated with 10 μg/mL PTX, which is similar to human therapeutic dose (6.7 mg/kg, Trental). However, in the pilot study to know the trends of protein expressions by PTX, 10 μg/mL PTX increased the expression of some proliferation-related proteins, RAS and NFkB signaling proteins, and even some inflammatory proteins in RAW 264.7 cells. These results were contrary to many reports insisting the anti-proliferative and anti-inflammatory effect of PTX. However, it was found that many experiments for PTX-induced effects on cells and animals were frequently performed by using higher dose of PTX, 100-500 μg/mL [5, 8, [19] [20] [21] [22] , than therapeutic dose of PTX, about 10 μg/mL. In order to elucidate the different pharmacological effect depending on the dose of PTX, the present study was performed to compare 10 μg/mL PTXinduced protein expressions with 300 μg/mL PTX-induced protein expression in RAW 264.7 cells.

As the essential protein signalings are intimately correlated and cross-talked with each other to maintain cellular homeostasis during proliferation, differentiation, inflammation, apoptosis, and senescence, it is necessary to know the global protein expression involving multiple signaling pathways in order to explain or predict the fate of cells involved with diseases or drug therapy. The present study examined PTX-induced global protein expression changes in RAW 264.7 cells through IP-HPLC, immunocytochemistry (ICC), and western blot. Particularly, IP-HPLC analysis is available to determine protein expression levels in different biological fluids, such as blood plasma, urine, saliva [23, 24] , inflammatory exudates [25] [26] [27] , cancer tissues [28, 29] , cell culture extract [30] [31] [32] [33] [34] , and blood plasma [32, 35] . Contrary to enzymelinked immunosorbent assay (ELISA). IP-HPLC uses protein A/G agarose beads in chaotic buffer solution and micro-sensitive UV spectroscopy to determine protein expression level

When approximately 70% confluent RAW 264.7 cells were spread over the surfaces of twowell culture slide dishes, the cells were treated with 10 μg/mL PTX for 12, 24, or 48 h, while the control cells were treated with 100 μL of normal saline. The cells on the culture slides were fixed with 4% paraformaldehyde solution for 20 min, permeabilized with cooled methanol for 10 min at -20˚C, and applied for immunohistochemistry using selected antisera (the same ones used in IP-HPLC, Table 1 ); Ki-67 for cellular proliferation, KMD4D and PCAF for epigenetic modification, TNFα, IL-6, TLR3, and TLR4 for inflammation, GSTO1/2, LC3, and GADD153 (CHOP) for endoplasmic reticulum stress, PARP-1 and caspase 3 for apoptosis, NSEγ for neural differentiation, MYH2 for muscular differentiation, TGF-β1, RUNX2, OPG, and BMPR2 for osteoblastic differentiation.

Immunocytochemical (ICC) staining was performed using the indirect triple sandwich method on the Vectastatin system (Vector Laboratories, USA), and visualized using a 3-amino-9-ethylcarbazole solution (Santa Cruz Biotechnology, USA) with no counter staining. The results were observed by optical microscope, and their characteristic images were captured (DP-73, Olympus Co., Japan) and illustrated. [38, 39] ; the number of antibodies overlapped; (). Abbreviations: AIF: Apoptosis inducing factor, AKAP13: A-kinase anchoring proteins 13, ALK1: Activin receptor-like kinase 1, ALP: Alkaline phosphatase, AMPK: AMP-activated protein kinase, pAKT: v-akt murine thymoma viral oncogene homolog, p-AKT1/2/3 phosphorylated (p-AKT, Thr 308), APAF1: Apoptotic protease-activating factor 1, APC: Adenomatous polyposis coli, ATF4: Activating transcription factor 4, ATM: Ataxia telangiectasia caused by mutations, AXIN2 (axis inhibition protein 2), BAD: BCL2 associated death promoter, BAK: BCL2 antagonist/killer, BAX: BCL2 associated X, : Nuclear factor of activated T-cells, cytoplasmic 1, NFkB: Nuclear factor kappa-light-chain-enhancer of activated B cells, NOS1: Nitric oxide synthase 1, NOXA: Phorbol-12-myristate-13-acetate-induced protein 1, NRAS: Neuroblastoma RAS Viral Oncogene homolog, NRF2: Nuclear factor (erythroid-derived)-like 2, OPG: Osteoprotegerin, PAI-1: Plasminogen activator inhibitor-1, PARP-1: Poly-ADP ribose polymerase 1, c-PARP-1: Cleaved-PARP-1, PCAF: p300/CBP-associated factor, PCNA: Proliferating cell nuclear antigen, PD-1 (CD279): Programmed cell death protein 1, PDGF-A: Plateletderived growth factor-A, PECAM-1 (CD31): Platelet endothelial cell adhesion molecule-1, PERK: Protein-like endoplasmic reticulum kinase, PGC-1α: Peroxisome proliferator-activated receptor gamma coactivator 1α, PI3K: Phosphatidylinositol-3-kinase, PIM-1: Proto-oncogene serine/threonine-protein kinase 1, PKC: Protein kinase C, PLCβ2: 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterse β2, PLK4: Polo like kinase 4 or serine/threonine-protein kinase, PSA: Prostate-specific antigen, PTEN: Phosphatase and tensin homolog, PUMA: p53 upregulated modulator of apoptosis, Rab1: RAS-related protein, Rab GTPases, RAF-B: v-Raf murine sarcoma viral oncogene homolog B, RANKL: Receptor activator of nuclear factor kappa-B ligand, Rb-1: Retinoblastoma-1, RUNX2: Runt-related transcription factor-2, SHH: Sonic hedgehog, SIRT3: Sirtuin 3 (silent mating type information regulation 2 homolog 3), NADdependent deacetylase, α-SMA: Alpha-smooth muscle actin, SMAD4: Mothers against decapentaplegic, drosophila homolog 4, SOD1: Superoxide dismutase-1, SOS1: Son of sevenless homolog 1, SOSTDC1: Sclerostin domain-containing protein 1, SOX9: SRY (sex-determining region Y)-related HMGbox transcription factor 9, SP1: Specificity protein 1, SRC1: Steroid receptor coactivator-1, STAT3: Signal transducer and activator of transcription-3, SVCT2: Sodium-dependent vitamin C transporter 2, TBX22: T-box transcription factor 22, TEM8: Tumor-specific endothelial marker 8 (anthrax toxin receptor 1), TERT: Human telomerase reverse transcriptase, TGase 2: Transglutaminase 2, TGF-β1: Transforming growth factor-β1, TNFα: Tumor necrosis factor-α, VCAM-1: Vascular cell adhesion molecule-1, VE-cadherin: Vascular endothelial cadherin, VEGF-A vascular endothelial growth factor A, VEGFR2: Vascular endothelial growth factor receptor 2, p-VEGFR2: Phosphorylated vascular endothelial growth factor receptor 2 (Y951), vWF: Von Willebrand factor, Wnt1: Proto-oncogene protein Wnt1, YAP1: Yes-associated protein 1.

https://doi.org/10.1371/journal.pone.0261797.t001

Some representative antisera were utilized for western blot analysis to assess the 10 μg/mL or 300 μg/mL PTX-induced protein expression in RAW 264.7 cells. Ki-67 was selected for cellular proliferation, p53, Rb1, and E2F1 for p53/Rb/E2F signaling, β-catenin, E-cadherin, VE-cadherin, Wnt1, and TCF1 for Wnt/β-catenin signaling, cMyc, MAX, and MAD1 for cMyc/ MAX/MAD network, KDM4D and HDAC10 for epigenetic modification, KRAS, HRAS, NRAS, ERK1, and p-ERK1 for RAS signaling, TNFα, TLR2, and TLR4 for inflammation, eIF2AK3, p-eIF2AK3, ATF4, LC3β, and c-caspase 3 for endoplasmic reticulum stress, TGF-β1, BMP2, RUNX2 for osteogenesis, NSEγ and NF1 for neural differentiation, and MYH2 and desmin for muscular differentiation. These antisera were the same ones used in IP-HPLC ( Table 1) .

The cells treated with 10 μg/mL and 300 μg/mL PTX for 0, 12, 24, and 48 h were collected with phosphate-buffered saline (PBS) separately, treated with trypsin-ethylene-diamine-tetraacetic acid (trypsin-EDTA) for one minute, and washed with PBS, and followed by cell lysis with ice-cold RIPA buffer (Sigma Aldrich, USA). The lysates were centrifuged at 12,000 g for 20 min at 4˚C. The protein concentration of the supernatant was quantified using a Bradford assay (BioRad, USA). Equal amounts (30 μg/lane) of the sample proteins were separated by 8, 10, 15, or 20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in Tris-glycine SDS running buffer (25 mM Tris, 0.1% SDS, and 0.2M glycine), and transferred to a nitrocellulose membrane. The membranes were blocked with 5% nonfat dry milk in TBST buffer (25 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.5) for 1 h. After washing three times with TBST buffer, the membrane was incubated with each primary antibody (dilution ratio = 1:1000, the same antibody used in IP-HPLC) and horseradish peroxidase-conjugated secondary antibody for 1 h separately. The protein bands were then detected using an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and digitally imaged using a ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, CA, USA). The level of β-actin expression was used as an internal control to normalize the expression of the target proteins. The size and intensity of protein bands from the cells treated with 10 μg/mL PTX for 0, 12, 24, and 48 h were demonstrated, and the protein bands from 10 μg/ mL PTX-treated cells were compared with those from 300 μg/mL PTX-treated cells.

Protein A/G agarose columns were separately pre-incubated with each 1 μg antibody for proliferation-related proteins (n = 13), cMyc/MAX/MAD network proteins (n = 6(3)), p53/Rb/ E2F signaling proteins (n = 7(3)), Wnt/β-catenin signaling proteins (n = 10), epigenetic modification proteins (n = 9), protein translation proteins (n = 8), growth factors (n = 23), RAS signaling proteins (n = 25), NFkB signaling proteins (n = 23(10)), upregulated inflammatory proteins (n = 34), downregulated inflammatory proteins (n = 30), p53-mediated apoptosis proteins (n = 15(1)), FAS-mediated apoptosis proteins (n = 10), protection and survival proteins (n = 30(7)), endoplasmic reticulum stress proteins (n = 19(11)), SHH and Notch signaling proteins (11(3)), cytodifferentiation proteins (n = 28(9)), neuromuscular differentiation proteins (19(10)), fibrosis proteins (19(16) ), oncogenesis proteins (n = 18(5)), angiogenesis proteins (n = 26(11)), osteogenesis proteins (n = 23(7)), and control housekeeping proteins (n = 3) (numbers in parenthesis indicate number of overlapping antibodies, Table 1 ). Although the immunoprecipitation is unable to define the size-dependent expression of target protein compared to western blot, it collects every protein containing a specific epitope against antibody. Therefore, the IP-HPLC can detect whole target proteins, precursor and modified ones, similar to enzyme-linked immunosorbent assay (ELISA).

The supernatant of the antibody-incubated column was removed, and followed by immunoprecipitation-based IP-HPLC. Briefly, each protein sample was mixed with 5 mL of binding buffer (150mM NaCl, 10mM Tris pH 7.4, 1mM EDTA, 1mM EGTA, 0.2mM sodium vanadate, 0.2mM PMSF and 0.5% NP-40) and incubated in the antibody-bound protein A/G agarose bead column on a rotating stirrer at room temperature for 1 h. After multiple washing of the columns with Tris-NaCl buffer, pH 7.5, in a graded NaCl concentration (0.15-0.3M), the target proteins were eluted with 300μL of IgG elution buffer (Pierce, USA). The immunoprecipitated proteins were analyzed using a precision HPLC unit (1100 series, Agilent, Santa Clara, CA, USA) equipped with a reverse-phase column and a micro-analytical UV detector system (SG Highteco, Hanam, Korea). Column elution was performed using 0.15M NaCl/20% acetonitrile solution at 0.5 mL/min for 15 min, 40˚C, and the proteins were detected using a UV spectrometer at 280 nm. The control and experimental samples were run sequentially to allow comparisons. For IP-HPLC, the whole protein peak areas (mAU � s) were obtained and calculated mathematically using an analytical algorithm (see S1 Fig) by subtracting the negative control antibody peak areas, and protein expression levels were compared and normalized using the square roots of protein peak areas. The ratios of the protein levels between the experimental and control groups were plotted into line and star graphs. Protein expressional changes of less than ±5%, ±5-10%, ±10-20%, or over ±20% changes were described as minimal, slight, significant, or marked, respectively [30] [31] [32] [33] 40] . The housekeeping proteins including β-actin, α-tubulin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were simultaneously used as internal controls.

In the previous study, the IP-HPLC results were compared with western blot data using cytoplasmic housekeeping protein (β-actin), the former showed minute error ranges less than ± 5% which were appropriate for statistical analysis, while the latter showed a large error range of more than 20% which were impossible to be analyzed statistically [40] (see S2 Fig) . Therefore, the present study mainly performed IP-HPLC, and its results were compared to representative findings of ICC and western blot performed with some selected antisera, even though ICC and western blot are usually involved with great error range (�20%).

The double IP-HPLC was designed to detect a protein complex or a binding body contained two different proteins. The first IP-HPLC was performed using the first antibody against one protein of complex as the above procedures to get 300 μL protein elute, which was applied as a protein sample in the second IP-HPLC. Subsequently, the second IP-HPLC was performed using the second column containing protein A/G beads bound with the second antibody against the other protein of complex. The protein elute sample was incubated with the protein A/G beads in the second column, and followed the same procedures of IP-HPLC described above.

As the overexpression and under-expression of essential 150 proteins observed in this study showed characteristics of 21 cellular functions affected by 10 μg/mL PTX. The maximum expression value (%) of upregulated proteins and the minimum protein expression values (%) of downregulated proteins at 12, 24, 48 h after 10 μg/mL PTX treatment were selected and plotted into a star graph.

Proportional data (%) of the experimental and control groups were plotted on line graphs and star plots. Their analyses were repeated two to six times until the standard deviations reached �±5%. The line graphs revealed time-dependent expression changes between the relevant proteins, and the star plots revealed the different expression levels of all proteins examined. The results were analyzed by measuring the standard error (s ¼ � ffi ffi ffiffi

). The expression of the control housekeeping proteins, i.e., β-actin, α-tubulin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was non-responsive (�5%) to 12, 24, or 48 h of PTX treatment [40, 41] (see S1 File).

10 μg/mL PTX-treated RAW 264.7 cells were evenly spread on two-well culture slide dishes and cultured for 48 h. Their monotonous small round nuclei were stained with hematoxylin, and then well distinguishable under microscope. They were increased in cell number depending on time, at 0, 12, 24, and 48 h (Fig 1A-1D ). 

The characteristic protein expressions were observed in RAW 264.7 cells through immunocytochemical (ICC) staining, that is, Ki-67, a marker of proliferation was strongly positive in 10 μg/mL PTX-treated cells at 12, 24, and 48 h compared to the untreated controls, and KDM4D and PCAF, markers of histone demethylation and acetylation, respectively, were strongly positive at 12 and 24 h. For the immunoreaction of inflammatory proteins, 10 μg/mL PTX-treated cells showed stronger positivity of TNFα, TLR3, and TLR4 at 24 and 48 h compared to the untreated controls, and increased positivity of IL-6 at 12, 24, and 48 h (Fig 2) . 10 μg/mL PTX-treated cells showed slight increase of immunoreaction for GSTO1/2 (a marker of antioxidant and cellular stress) and LC3β (a marker of autophagosome biogenesis), compared to the untreated controls. Caspase 3, a marker of apoptosis executioner was markedly positive in 10 μg/mL PTX-treated cells at 12, 24, and 48 h, while the immunoreaction of GADD153 (CHOP, a marker of endoplasmic reticulum stress) and PARP-1 (a marker of DNA damage) was almost similar in the experimental and control cells (Fig 3) .

10 μg/mL PTX-treated cells showed slight increase of NSEγ immunoreaction at 24 and 48 h compared to the untreated controls, and marked increase of MYH2 (a marker of muscular differentiation) immunoreaction at 12, 24, and 48 h. Regarding the markers of osteoblastic differentiation, RUNX2, OPG, BMPR2, and TGF-β1 were markedly positive in 10 μg/mL PTXtreated cells at 12, 24, and 48 h (Fig 4) . These results indicate 10 μg/mL PTX affect RAW 264.7 cells to have a potential for neuro-muscular and osteogenic differentiation.

For the proteins relevant to proliferation, 10 μg/mL PTX-treated cells showed stronger bands for markers of cell proliferation (Ki-67), p53/Rb signaling (p53, Rb1, and E2F1), Wnt/β-catenin signaling (Wnt1, β-catenin, TCF1), and guided cell migration (E-cadherin and VE- 

cadherin) at 12, 24, and 48 h than the untreated controls. Among the cMyc/MAX/MAD network proteins, the bands of cMyc and MAX were gradually attenuated at 12, 24, and 48 h, while the bands of MAD1 were increased. The proteins relevant to epigenetic modification, KDM4D and HDAC10 were increased in 10 μg/mL PTX-treated cells at 12, 24, and 48 h versus the untreated controls. RAS signaling proteins, KRAS, HRAS, NRAS, ERK1, and p-ERK1 were also increased at 12, 24, and 48 h (Fig 5) .

TNFα, an inflammatory cytokine, TLR2 and TLR4, markers of innate immunity were increased in 10 μg/mL PTX-treated cells at 12, 24, and 48 h compared to the untreated controls. ER stress proteins, eIF2AK3 and p-eIF2AK3, a marker for autophagy formation, LC3β, and an apoptosis executing protein, caspase 3 were coincidently increased at 24 and 48 h. 10 μg/mL PTX-treated cell showed stronger bands of osteoblastic differentiation proteins, TGF-β1, BMP2, RUNX2, and ATF4 than the untreated controls, and slightly strong bands of nerve differentiation proteins, NSEγ and NF1, and muscle differentiation proteins, MYH2 and desmin at 12, 24, and 48 h compared to the untreated controls ( Fig 6) .

Additionally, 300 μg/mL PTX-induced protein expressions in RAW 264.7 cells were also explored by western blot, and compared with 10 μg/mL PTX-induced protein expressions. 300 μg/mL PTX slightly decreased the expression of Ki-67, cMyc, MAX, E2F1, Wnt1, and 

TCF1 at 12, 24, and 48 h versus the untreated controls, while 10 μg/mL PTX increased the protein expressions of Ki-67, E2F1, Wnt1, and TCF1, and decreased the protein expressions of cMyc and MAX. The expression of HDAC10 was gradually decreased by 300 μg/mL PTX at 12, 24, and 48 h, while slightly increased by 10 μg/mL PTX (Fig 7) .

Regarding RAS signaling, KRAS, ERK1, and pERK1 were slightly downregulated by 300 μg/mL PTX at 12, 24, and 48 h, while upregulated by 10 μg/mL PTX. Apoptosis proteins, 

p53 and c-caspase 3 were rarely affected by 300 μg/mL PTX, while 10 μg/mL PTX slightly decreased the p53 expression at 12, 24, and 48 h but increased the c-caspase 3 expression at 12, 24, and 48 h. The ER stress proteins, eIF2AK3 and p-eIF2AK3 were slightly upregulated by 300 μg/mL PTX, while eIF2AK3 and p-eIF2AK3 were slightly downregulated but ATF4 was upregulated by 10 μg/mL PTX (Fig 7) .

The expressions of inflammatory proteins, TNFα and TLR2 were rarely affected by 300 μg/ mL PTX compared to the untreated controls, while increased by 10 μg/mL PTX at 12, 24, and 48 h. And the expressions of osteogenesis proteins, BMP2, RUNX2, and ATF4 were slightly decreased by 300 μg/mL PTX, while increased by 10 μg/mL PTX at 12, 24, and 48 h. On the other hand, the TGF-β1 expression was rarely affected by 300 μg/mL PTX, while slightly increased by 10 μg/mL PTX at 12 and 24 h. And the expression of house-keeping protein, βactin was almost not affected by 10 μg/mL and 300 μg/mL PTX at 12, 24, and 48 h (Fig 7) .

10 μg/mL PTX-treated RAW 264.7 cells were extensively explored for different protein expression by IP-HPLC using 409 antisera, and 300 μg/mL PTX-treated cells were simply done using 

Effects of 10 μg/mL PTX on the expression of proliferation-related proteins. RAW 264.7 cells treated with 10 μg/mL PTX for 12, 24, or 48 h showed significant increases in the expression of proliferation-activating proteins including Ki-67 (by 7.7% at 48h), proliferating cell nuclear antigen (PCNA, 7.3% at 48 h), polo-like kinase 4 (PLK4, a regulator of centriole duplication, 12.1% at 48h), CDK4 (4.2% at 12h), cyclin D2 (a regulator of cyclin-dependent kinase, 10.1% at 12 h), cell division cycle 25A (cdc25A, 6.2% at 12h), transcription factor BRG1 (ATP-dependent chromatin remodeler SMARCA4, 4% at 24h), and reactive increase in the expression of p14ARF (an alternate reading frame (ARF) protein product of the CDKN2A locus, 17.2% at 24 h), p15/16INK (inhibitors of cyclin-dependent kinases (INK), 13% at 48 h), and p21CIP1 (a CDK-interacting protein 1 (CIP1), 5.2% at 48 h) versus the untreated controls. On the other hand, the expressions of mitosis phase promoting factor (MPF) recognized by a mitosis-specific monoclonal antibody 2 (MPM2) and lamin A/C involved in nuclear stability, chromatin structure and gene expression were decreased by 6.7% and 12.1% at 12 h, respectively, and the expression of p27KIP1 (a cyclin dependent kinase inhibitor protein 1 (KIP1)), was minimally affected by PTX (�5%) (Fig 8A and 8B) .

Effects of 10 μg/mL PTX on the expression of cMyc/MAX/MAD network proteins. The expression of cMyc (regulator genes and proto-oncogenes that code for transcription factors) was increased by 8.2% at 12h after 10 μg/mL PTX treatment but gradually decreased to the untreated control level at 48 h, the expression of MAX (bHLH-Zip protein forming heterodimer with cMyc) was decreased by 3% at 48 h, while the expression of MAD1 (bHLH-Zip protein forming heterodimer with MAX which can oppose functions of Myc-MAX heterodimers) was increased by 4.8% at 48 h versus the untreated controls (Fig 8C and 8D ). Whereas the double IP-HPLC using first antibody of cMyc and second antibody of MAX or MAD1 showed that the heterodimers of cMyc and MAX were increased by 11% at 24 h and 11.9% at 48 h, while the heterodimer of cMyc and MAD1 was decreased by 4.2% at 12 h and 2.1% at 24 h compared to the untreated controls (Fig 8E and 8F ). On the other hand, the expressions of cMyc/MAX/MAD network interacting proteins, CDK4 and cyclin D2 were increased by 4.2% at 12 h and 10.1% at 12 h, respectively, but the expression of p27 (cyclin-dependent kinase inhibitor 1B) was minimally affected by PTX (�5%) (Fig 8C and 8D ).

In the double IP-HPLC using antisera of cMyc/MAX and cMyc/MAD1, the cMyc-MAX heterodimer was increased by 11% at 24 h and 11.9% at 48 H, while the cMyc-MAD heterodimer was decreased by 4.2% at 12 h and 2.1% at 24 h compared to the untreated controls. On the other hand, CDK4-p27 complex was consistently reduced by 4.6%, at 12 h, 4.1% at 24 h, and 4.7% at 48h in the double IP-HPLC using CDK4 and p27 antisera (Fig 8E and 8F) .

Effects of 10 μg/mL PTX on the expression of p53/Rb/E2F signaling proteins. 10 μg/ mL PTX decreased the expression of tumor suppressor proteins, that is, p53 by 17.5% at 24 h, Rb1 by 9.8% at 48 h, and phosphorylated Rb1 (p-Rb1) at 5.5% in RAW 264.7 cells versus the untreated controls, while the expression of objective transcription factor, E2F1 was increased by 10.6% at 12 h. And the Ser/Thr-kinase components of cyclin D2 and CDK4 were upregulated by 10.1% and 4.2% at 12 h, respectively, and a cyclin-dependent kinase inhibitor protein, p21CIP1 was also upregulated by 5.2% at 48 h (Fig 9A and 9B ).

In the double IP-HPLC using antisera of E2F1/Rb1, CDK4/p21, and E2F1/VE-cadherin, the E2F1-Rb1 and CDK4-p21 complexes were increased by 8.4% and 12.9% at 24 H, respectively, but E2F1-VE-cadherin complex detection as a negative control was minimally affected by 10 μg/mL PTX (Fig 9C and 9D ). On the other hand, CDK4-p27 complex was consistently reduced by 4.6% at 12 h, 4.1% at 24 h, and 4.7% at 48h in the double IP-HPLC using CDK4 and p27 antisera (Fig 8E and 8F) .

Effects of 10 μg/mL PTX on the expression of Wnt/β-catenin signaling proteins. 10 μg/ mL PTX increased the protein expressions of Wnt1 (by 13% at 24 h), β-catenin (by 18.5% at 24 h), adenomatous polyposis coli (APC, by 13.8% at 48 h), snail (by 13.1% at 48 h), and T-cell factor 1 (TCF1, a transcription factor, by 13.4% at 12 h) versus the untreated controls, while decreased the protein expression of snail (a transcription factor for the repression of adhesion molecule E-cadherin) by 8% at 12 h and AXIN2 (a regulator of β-catenin stability) by 8.7% at 24 h. On the other hand, E-cadherin (a type-I transmembrane protein stabilized by β-catenin) and VE-cadherin (vascular endothelial cadherin) were increased by 17.4% at 48 h and 21.9% at 12 h, respectively, while the expressions of Wnt signaling cofactors, MMP9 and vimentin which are necessary in the process of epithelial-mesenchymal transition, were decreased by 10.4% and 12.9% at 24 h, respectively (Fig 9E and 9F) .

These results indicate Wnt/β-catenin signaling was enhanced with concomitant upregulation of Wnt1, β-catenin, APC, and TCF1 by 10 μg/mL PTX, but the activation of Wnt/β-catenin signaling was not followed by upregulation of snail, AXIN2, MMP9, and vimentin, but led to the overexpression of E-cadherin and VE-cadherin as shown in the results of double IP-HPLC (Fig 9G and 9H) .

Effects of 10 μg/mL PTX on the expression of epigenetic modification proteins. 10 μg/ mL PTX increased the expression of histone H1 (by 10.6% at 24 h), lysine-specific demethylase 4D (KDM4D, 12% at 48 h), high mobility group box 1 (HMGB1, 8.8% at 24 h), and p300/ CBP-associated factor K (lysine) acetyltransferase 2B which has histone acetyl transferase activity (PCAF, by 14% at 12 h) versus the untreated controls, while decreased the expression of histone deacetylase 10 (HDAC10, 9.6% at 24 h), DNA (cytosine-5)-methyltransferase 1 (DNMT1: 23.5% at 48 h), DNA methyltransferase 1-associated protein 1 (DMAP1: 17.7% at 48 h), histone-lysine N-methyltransferase enzyme (enhancer of zeste homolog 2 (EZH2), 6.6% at 24 h), and methyl-CpG binding domain 4 (MBD4: 6.7% at 12 h) (Fig 10A and 10B) .

Effects of 10 μg/mL PTX on the expression of protein translation proteins. RAW 264.7 cells treated with 10 μg/mL PTX showed increase in the expression of protein translation protein: deoxyhypusine hydroxylase (DOHH, by 7.6% at 12 h), deoxyhypusine protein synthase (DHPS, 17.1% at 24 h), eukaryotic translation initiation factor 5A1 (eIF5A1, 6.5% at 12 h), and eIF5A2 (7.5% at 24 h) versus the untreated controls. On the other hand, the essential factor for protein synthesis to form a ternary complex (TC) with GTP and the initiator Met-tRNA, that is, eIF2α and p-eIF2α were decreased by 8.7% at 12 h and 5.7% at 48 h, respectively, and eukaryotic translation initiation factor 2-α kinase 3 (eIF2AK3, a protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK)) was decreased by 9.3% at 12 h, but p-eIF2AK3 was reactively increased by 6.7% at 48 h (Fig 10C and 10D) .

Effects of 10 μg/mL PTX on the expression of growth factor. RAW 264.7 cells after 10 μg/mL PTX administration showed marked decrease in the expression of hepatocyte growth factor receptor α (HGFα, by 17.5% at 24 h), Met (HGF receptor, 9.9% at 48 h), fibroblast growth factor-1 (FGF1, 5.5% at 12 h), FGF2 (10.3% at 12 h), FGF7 (2% at 48 h), connective tissue growth factor (CTGF, 17.7% at 24 h), and estrogen receptor-β (ERβ, 14.1% at 24 h) versus the untreated controls. Particularly, PTX decreased the expression of TGF-α (by 21% at 48 h), TGF-β1 (12.5% at 12 h), TGF-β2 (18.5% at 48 h), TGF-β3 (16.4% at 24 h), activin receptor-like kinase 1 (ALK1, 14.6% at 12 h), SMAD2/3 (15.8% at 12 h), and HER1 (epidermal growth factor receptor, 13.4%), while compensatory increased the expression of SMAD4 (14.9% at 48 h), p-SMAD4 (7.1% at 24 h), growth hormone (GH, 11.4% at 48 h), growth hormone releasing hormone (GHRH, 16.9% at48 h), insulin-like growth factor 2 receptor (IGF2R, 11.4% at 48 h), HER2 (EGF receptor tyrosine-protein kinase erbB-2, 9.6% at 24 h), and epidermal growth factor (EGF, 18.8% at 24 h). On the other hand, the expressions of FGF7, IGF1, and PDGF-A were affected minimally by PTX (�5%) (Fig 10E and 10F) .

Effects of 10 μg/mL PTX on the protein expressions of RAS signaling proteins. 10 μg/ mL PTX affected RAS signaling protein expressions of RAW 264.7 cells positively or negatively. The RAS signaling proteins were initially upregulated at 12 Whereas the expressions of other RAS signaling proteins were decreased, that is, c-Jun Nterminal kinase-1 (JNK1) by 5.6% at 24 h, phosphorylated JNK1 (p-JNK1, Thr 183/Tyr 185) by 11.1% at 24 h, MEK kinase 1 (also designated MAP kinase kinase kinase 1, MKKK1, MAP3K1 or MEKK1) by 11.7% at 12 h, mammalian target of rapamycin (mTOR) by 19.5% at 24 h, phosphorylated mTOR (p-mTOR) by 15.7% at 48 h, Janus kinase 2 (JAK2, non-receptor tyrosine kinase) by 14.8% at 48 h, and son of sevenless homolog 1 (SOS1) by 22.3% at 12 h. On the other hand, the expression of phosphatase and tensin homolog (PTEN) was affected minimally by PTX (�5%) (Fig 11A and 11B) .

Effects of 10 μg/mL PTX on the expression of NFkB signaling proteins. 10 μg/mL PTX had different effects on the expression of NFkB signaling proteins in RAW 264.7 cells. PTX markedly upregulated nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) by 20.7% at 24 h but slightly downregulated ikappa B kinase (IKK) by 6.8% at 12 h versus the untreated controls, and subsequently increased the expression of downstream effector proteins of NFkB signaling, that is, p38 mitogen-activated protein kinase (p38) by 13.1% at 48 h, phosphorylated p38 (p-p38) by 12.3% at 48 h, growth arrest and DNA damage 45 (GADD45) by 18.7% at 48 h, multiple drug resistance (MDR) by 12.5% at 48 h, protein kinase C (PKC) by 8.6% at 24 h, p-PKC1α by 11.5% at 24 h, steroid receptor co-activator-1 (SRC1) by 12.3% at 48 h, and A-kinase anchoring proteins (AKAP13) by 16.4% at 24 h.

On the other hand, the expressions of some downstream regulating proteins of NFkB signaling were decreased, that is, mTOR by 19.5% at 24 h, p-mTOR by 15.7% at 48 h, and peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α) by 12.1% at 24 h, MEKK1 by 11.7% at 12 h, JAK2 by 14.8% at 48 h, and nuclear factor of activated T-cells 1 (NFATC1) by 7.3% at 24 h. The expressions of GADD153, nuclear factor (erythroid-derived 2)-like 2 (NRF2), 5' AMP-activated protein kinase α (AMPKα), and nuclear factor of activated T cells (NFAT5) were only minimally affected by PTX (� 5%) (Fig 11C and 11D ).

Inflammatory proteins upregulated by 10 μg/mL PTX. Among the inflammatory proteins, some proteins were upregulated by 10 μg/mL PTX in RAW 264.7 cells as follows, that is, tumor necrosis factor α (TNFα) by 10.6% at 24 h, interleukin-1 (IL-1) by 5 On the other hand, the expression of MMP12, TLR2, TLR7, and C-reactive protein (CRP) showed a trend of increase but were only minimally affected by PTX (� 5%) (Fig 11E and 11F ). 19 .5% at 24 h. On the other hand, the expression of CD40 and programmed cell death protein 1/1 (PD-1, CD279) showed a trend of decrease but were only minimally affected by PTX (� 5%) (Fig 12A and 12B) . 

Effects of 10 μg/mL PTX on the expression of p53-mediated apoptosis proteins. 10 μg/ mL PTX significantly reduced the expression of p53-mediated pro-apoptotic proteins, that is, p53 by 17.5% at 24 h, BCL2 homologous antagonist/killer (BAK) by 5.5% at 48 h, p73 by 23.9% at 12 h, NOXA (a pro-apoptotic member of the BCL2 protein family) by 7.8% at 24 h, p53 upregulated modulator of apoptosis (PUMA, mitochondria pro-apoptotic BCL-2 homolog) by 6.1% at 24 h, apoptosis regulator BAX by 24% at 48 h, apoptotic protease activating factor 1 (APAF1) by 10.9% at 48 h, caspase 9 by 14.4% at 12 h, and c-caspase 9 by 15.7% at 24 h, apoptosis inducing factor (AIF) by 10.9% at 48 h, poly [ADP-ribose] polymerase 1 (PARP1) by 13.1% at 12 h, and cleaved PARP (c-PARP) by 21.5% at 12 h versus the untreated controls, while it increased the expression of murine double minute-2 homolog (MDM2, negative regulator of p53) by 8.9% at 12 h and minimally affected the expression of B cell lymphoma 2 (BCL2, anti-apoptotic protein) (� 5%) (Fig 12C and 12D) .

Effects of 10 μg/mL PTX on the expression of FAS-mediated apoptosis proteins. RAW 264.7 cells treated with 10 μg/mL PTX showed increases in the expression of FAS-mediated apoptosis proteins, that is, FAS ligand (FASL) by 20 (Fig 12E and 12F) .

Effects of 10 μg/mL PTX on the expression of protection-related proteins. 10 μg/mL PTX-treated RAW 264.7 cells showed increases in the expression of cellular stress protectionand antioxidant-related proteins versus the untreated controls, as follows: heat shock protein-70 (HSP70) by 17.2% at 12 h, Cu-Zn superoxide dismutase-1 (SOD1) by 28.2% at 48 h, glutathione S-transferase ω 1/2 (GSTO1/2, a detoxifying enzyme) by 30.3% at 48 h, nitric oxide synthases-1 (NOS1) by 6.4% at 12 h, LC3β by 11.6% at 48 h, sodium-dependent vitamin C transporter 2 (SVCT2) by 6.6% at 48 h, a transcription factor regulating the expression of antioxidant proteins NRF2 by 4.1% at 24 h, and energy expenditure hormone leptin by 13.1% at 24 h versus the untreated controls. Whereas PTX decreased the expression of cellular maintenance proteins: heme oxygenase-1 (HO1) by 12.1% at 48 h, small heat shock protein HSP27 by 14.2% at 48 h, HSP90α/β by 15.8% at 24 h, mTOR by 19.5% at 24 h, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) by 12.1% at 24 h. The expressions of 5' AMP-activated protein kinase α (AMPKα, an enzyme that regulates cellular energy homeostasis) was only minimally affected by PTX (� 5%) (Fig 13A and 13B) .

Effects of 10 μg/mL PTX on the expression of survival and aging-related proteins. 10 μg/mL PTX-treated RAW 264.7 cells showed increases in the expression of survival and aging-related proteins, that is, pAKT1/2/3 by 6.5% at 24h, PKC by 8.6% at 24h, pPKC1α by 11.5% at 24h, SP-1 by 8.3% at 48h, a stress responsive deacetylase sirtuin 6 by 5.8% at 24 h, a master regulator of the regulatory pathway in the development and function of regulatory T cells FOXP3 by 7.4% at 12h, a type-I membrane protein related to β-glucuronidase Klotho by 7.1% at 12h, and PLCβ2 by 13.1% at 48h versus the untreated controls. Whereas PTX decreased the expression of telomere reverse transcriptase (TERT) by 10.4% at 12h, SP3 by 16.4% at 48h, sirtuin 1 by 9% at 24h, and FOXP4 by 11% at 48h. The expressions of sirtuin 7, FOXO1 (a transcription factor negatively regulating adipogenesis), and FOXO3 were only minimally affected by PTX (� 5%) (Fig 13C and 13D) .

Effects of 10 μg/mL PTX on the expression of endoplasmic reticulum stress proteins. 10 μg/mL PTX had different effects on the expression of endoplasmic reticulum stress proteins in RAW 264.7 cells. PTX downregulated the proteins contributing to ER stress signaling; eIF2AK3, (PERK, which functions as an ER kinase) by 9.3% at 12 h, eIF2α and p- eIF2α (essential factors for protein synthesis also responsible for ER stresses) by 8.7% at 12 h and 5.7% at 48 h, respectively, activating transcription factor 6α (ATF6α) by 9% at 12 h, binding immunoglobulin protein (BIP, a HSP70 molecular chaperone) by 8.4% at 12 h, serine/threonine-protein kinase/endoribonuclease inositol-requiring enzyme 1 α (IRE1α) by 8.2% at 24 h, PGC-1α by 12.1% at 24 h, caveolin-1 by 8.3% at 48 h, and AIF by 10.9% at 48 h compared to the untreated controls. On the other hand, PTX upregulated the proteins contributing to ERstress environment in cells; HSP70 by 17.2% at 12 h, p-eIF2AK3 by 6.7% at 48 h, ATF4 (cAMP-response element binding protein 2) by 8% at 24 h, AP1M1 (the medium chain of the trans-Golgi network clathrin-associated protein complex AP-1) by 12.9% at 48 h, calnexin (a chaperone for the protein folding in the membrane of the ER) by 8.5% at 12 h, LC3β (microtubule-associated proteins 1A/1B light chain 3B contributing to autophagosome biogenesis) by 11.6% at 48 h, and endothelin-1 (inducing Ca 2+ release from ER) by 17.8% at 24 h. The expressions of GADD153 (C/EBP homologous protein (CHOP)) and p-GADD153 were only minimally affected by PTX (� 5%) (Fig 13E and 13F) .

Effects of 10 μg/mL PTX on the expression of SHH/PTCH/GLI and Notch/Jagged signaling proteins. 10 μg/mL PTX was found to influence the expression of SHH/PTCH/GLI signaling proteins positively or negatively in RAW 264.7 cells. PTX upregulated the upstream proteins of SHH/PTCH/GLI signaling; sonic hedgehog (SHH) by 14.6% at 24 h, patched homolog 1 (PTCH1, the receptor for sonic hedgehog) by 8.8% at 48 h, and CD44 (HCAM, the activator of SHH signaling) by 9.8% at 48 h versus the untreated controls, while downregulated the downstream proteins of SHH/PTCH/GLI signaling; GLI1(Glioma-associated oncogene, the effectors of SHH signaling) by 12.9% at 24 h, EpCAM (epithelial cell adhesion molecule, involved in SHH signaling) by 3.1% at 48 h, and BCL2 (GLI binding site in BCL2 promoter, upregulated by GLI1) by 4.6% at 12 h.

On the other hand, PTX downregulated Notch/Jagged signaling proteins; Notch1 (Notch homolog 1, translocation-associated (Drosophila)) by 5.3% at 48 h, Jagged2 (ligand for Notch) by 6.1% at 24 h, and VEGF-A (crosstalk between VEGF and Notch signaling) by 6.7% at 48 h. The expressions of Notch upstream signaling proteins, HIF1α (hypoxia-inducible factors 1α) and Wnt1 were compensatory increased by 13% and 13% at 24 h, respectively (Fig 13G and 13H) .

Effects of 10 μg/mL PTX on the expression of cytodifferentiation proteins. 10 μg/mL PTX was found to influence the expression of cytodifferentiation proteins positively or negatively in RAW 264.7 cells. PTX upregulated some cytodifferentiation proteins, that is, α-actin by 12% at 24 h, transglutaminase 2 (TGase 2) by 15.3% at 48 h, protein kinase C (PKC, serine/ threonine protein kinase, 8.6% at 24 h), p-PKC1α(11.5% at 24 h), focal adhesion kinase (FAK, 6.3% at 48 h), A-kinase anchoring proteins 13 (AKAP13, 7.8% at 48 h), calmodulin (CaM, the universal calcium sensor) by 8.2% at 48 h, calnexin by 8.5% at 12 h, cystatin A (a thiol protease inhibitor) by 6.9% at 12 h, SOX9 (SRY-related HMG-box) by 9.6% at 48 h, AP-1 complex subunit mu-1 (AP1M1, localized at Golgi vesicles for endocytosis and Golgi processing) by 12.9% at 48 h), phosphoinositide-specific phospholipase C β2 (PLCβ2) by 13.1% at 48 h, leucine-rich repeat-containing G-protein coupled receptor 4 (LGR4) by 6.4%, E-cadherin by 17 On the other hand, the expressions of cysteine-rich protein that participates in cytoskeletal remodeling (CRIP1) by 16.3% at 12 h), TBX22 (T-box transcription factor), and epithelial cell adhesion molecule (EpCAM) were only minimally affected by PTX (� 5%) ( Fig  14A and 14B) .

Effects of 10 μg/mL PTX on the expression of neuromuscular differentiation proteins. 10 μg/mL PTX was found to have positive influence on the expression of neuromuscular differentiation proteins in RAW 264.7 cells. PTX upregulated some neuromuscular differentiation proteins, that is, neuron specific γ enolase (NSEγ) by 13.6% at 24 h, glial fibrillary acidic protein (GFAP) by 9.5% at 12h, myosin heavy chain 2 (MYH2) by 28.6% at 24 h, desmin by 12.2% at 12h, calmodulin (CaM) by 8.2% at 48 h, calnexin by 8.5% at 12 h, cystatin A by 6.9% at 12 h, AP-1 complex subunit mu-1 (AP1M1) by 12.9% at 48 h, focal adhesion 

kinase (FAK, substrate for tyrosine kinase of Src) by 6.3% at 48 h, SHH by 14.6 at 24 h, β-catenin by 18.5% at 24 h, Wnt1 by 13% at 24 h, TGase 2 by 15.3% at 48 h, and α-smooth muscle actin (α-SMA) by 30.3% at 12 h, while downregulated other neuromuscular differentiation proteins, that is, S-100 by 13.8% at 48 h, unconventional myosin-1a (membrane binding class I myosin), 10.9% at 12 h, neurofibromin 1 (NF1) by 6.3% at 12 h. The expressions of neurokinin 1 receptor (NK1R, substance P receptor) and cysteine-rich protein 1 (CRIP1) were only minimally affected by PTX (� 5%) (Fig 14C and 14D) .

Effects of 10 μg/mL PTX on the expression of fibrosis proteins. 10 μg/mL PTX was found to decrease the expression of fibrosis-inducing proteins; FGF1 by 5.5% at 12 h, FGF2 by 10.3% at 12 h, TGF-β1 by 12.5% at 12 h, CTGF by 17.7% at 12 h, collagen 3A1 by 13.7% at 48 h, collagen 4 by 14.6% at 24 h, collagen 5A by 6.9% at 24 h, laminin α5 by 14.2% at 48 h, integrin β1 by 8.7% at 12 h, plasminogen activator inhibitor-1 (PAI1) by 8.7% at 12 h, α1antitrypsin by 7% at 24 h, elafin (peptidase inhibitor 3, elastase-specific protease inhibitor) by 5.2% at 48 h, and also to increase anti-fibrosis proteins; plasminogen by 15.6% at 48 h, integrin α2 by 16% at 24 h, integrin α5 by 12.2% at 48 h, and capillary morphogenesis gene 2 (CMG2) by 13.5% at 12 h. On the other hand, the expression of endothelin-1, a key role of vascular homeostasis, was reactively upregulated by 17.8% at 24 h. And the expressions of FGF7 and platelet-derived growth factor A (PDGF-A) were only minimally affected by PTX (� 5%) (Fig 14E and 14F) .

Effects of 10 μg/mL PTX on the expression of oncogenesis proteins. 10 μg/mL PTX was found to influence the expression of oncogenesis proteins positively or negatively in RAW 264.7 cells. PTX decreased the expression of tumor suppressor proteins; breast cancer type 1 susceptibility protein (BRCA1) by 8.4% at 12 h, breast cancer type 2 susceptibility protein (BRCA2) by 18.6% at 24 h, neurofibromin 1 (NF1, a GTPase-activating protein that negatively regulates RAS/MAPK pathway activity) by 6.3% at 12 h, ataxia telangiectasia caused by mutations (ATM, a serine/threonine protein kinase recruited and activated by DNA double-strand breaks) by 13.9% at 24 h, maspin (a mammary serine protease inhibitor, serpin superfamily) by 9.5% at 24 h, deleted in malignant brain tumors 1 protein (DMBT1, a glycoprotein that interacts between tumor cells and the immune system) by 18% at 24 h, methyl-CpG-binding domain protein 4 (MBD4, a DNA repair enzyme that removes mismatched U or T) by 6.7% at 12 h, p53 by 17.5% at 24 h, retinoblastoma protein (Rb1) by 9.8% at 48 h, but increased the expression of PTCH1 (Protein patched homolog 1, a suppressor of smoothened release, which signals cell proliferation) by 10.1% at 24 h. On the other hand, PTX increased the expression of oncogenic proteins; carcinoembryonic antigen (CEA) by 9.6% at 12 h, 14-3-3 θ proteins (a phosphoserine binding protein that regulates Cdc25C) by 10.9% at 48 h, survivin (a negative regulator of apoptosis) by 11.9% at 48 h, mucin 4 (an anti-adhesive glycoprotein that contributes to tumor development and metastasis) by 15.6% at 24 h, Yes-associated protein 1 (YAP1, a potent oncogene that binds to 14-3-3) by 10.3% at 12 h, but decreased the expression of mucin 1 (a glycoprotein with extensive O-linked glycosylation of its extracellular domain, oncogenic epithelial membrane antigen) by 21.2% at 48 h. the expression of phosphatase and tensin homolog (PTEN, tumor suppressor protein) and PIM1 (proto-oncogene serine/threonine-protein kinase) were only minimally affected by PTX (� 5%) (Fig 15A and 15B) .

Effects of 10 μg/mL PTX on the expression of angiogenesis proteins. 10 μg/mL PTX reduced the expression of major angiogenesis proteins in RAW 264. 

adhesion molecule (HCAM)) by 9.8% at 24 h. The expression of PDGF-A and CD31 (platelet endothelial cell adhesion molecule (PECAM-1)) were only minimally affected by PTX (� 5%) (Fig 15C and 15D) .

Effects of 10 μg/mL PTX on the expression of osteogenesis proteins. 10 μg/mL PTXtreated RAW 264.7 cells showed increase in the expression of osteogenesis proteins, that is, bone morphogenetic protein-2 (BMP2, 16 activator of nuclear factor kappa-Β ligand (RANKL, a binding partner of OPG) by 6.6% at 12 h, TGF-β1 by 12.5% at 12 h, connective tissue growth factor (CTGF, CCN2, a role in chondrogenesis and angiogenesis) by 17.7% at 12 h, cathepsin K (a lysosomal cysteine protease involved in bone remodeling and resorption [46] ) by 9.2% at 48 h, HSP90α/β (a crucial regulator of vesicular transport of cellular proteins in osteoclasts [47] ) by 16.1% at 24 h, and sclerostin domain-containing protein 1 (SOSTDC1, a bone morphogenetic protein antagonist) by 7% at 24 h (Fig 15E and 15F ).

Global protein expressions in 10 μg/mL PTX-treated RAW 264.7 cells Fig 16 presents the global protein expression changes in 150 representative proteins of 21 different protein signaling pathways as a star plot. 10 μg/mL PTX was found to affect the expression of proteins in different signaling pathways of RAW 264.7 cells, and regulated characteristic cellular functions. PTX increased cell proliferation by upregulating proliferation-activating proteins, Ki-67, PCNA, cyclin D2, and CDK4, and enhancing proliferationrelated pathways; that is, cMyc/MAX/MAD network by upregulating cMyc and MAX, p53/ Rb/E2F by upregulating E2F1 and downregulating p-Rb1, and Wnt/β-catenin signaling by upregulating Wnt1, APC, β-catenin, TCF1 and by downregulating snail and AXIN2. PTX decreased histone/DNA methylation by downregulating EZH2, DNMT1, and DMAP1 and upregulating KDM4D, increased histone acetylation by upregulating PCAF and HMGB1, and downregulating HDAC-10, and also enhanced protein translation by upregulating DOHH, DHS, and eIF5A1.

PTX was found to downregulate TGF-β1, TGF-β2, TGF-β3, SMAD2/3, HGFα, Met, but reactively upregulate SMAD4, while it stimulated RAS signaling by upregulating KRAS, HRAS, pAKT1/2/3, PI3K, Rab1, ERK1, and pERK1 and downregulating JAK2, p-JNK1, and MEKK1, and simultaneously elevated NFkB signaling by upregulating NFkB, GADD45, p38, p-p38, and MDR, and downregulating p-mTOR and PGC1α.

PTX increased the expression of acute inflammatory proteins (TNFα, IL-1, IL-6, granzyme B, MCP1, CXCR4, CTLA4, M-CSF, and MMP1), cell-mediated immunity proteins (CD4, CD8, CD80, HLA-DR, and perforin), and innate immunity proteins (lactoferrin, versican [48] , TLR3, and TLR4).

PTX diminished p53-mediated apoptosis by downregulating p53, BAD, NOXA, APAF1, caspase 9, c-caspase 9, and c-PARP in RAW 264.7 cells, whereas it enhanced FAS-mediated apoptosis by upregulating FAS, FADD, caspase 7, c-caspase 8, c-caspase 10, caspase 3, and ccaspase 3. On the other hand, PTX activated cell protection by increasing the expression of HSP70, SOD1, GSTO1/2, SVCT2, NRF2, and NOS1 [49] , and stimulated cell survival and homeostasis by upregulating PLCβ2, SP1, and downregulating TERT, SIRT1, SP3, AMPKα, and PGC1α. Particularly, PTX attenuated ER-stress by downregulating HSP27, IRE1α, eIF2AK3, p-eIF2α, ATF6α, p-GADD153, caveolin-1, AIF, BIP, and upregulating LC3β. 10 μg/mL PTX activated the growth factors, epigenetic modification, protein translation, RAS and NFkB signaling, protection, neuromuscular ad osteoblastic differentiation, acute inflammation associated with innate immunity and cell-mediated immunity, but inactivated ER stress, fibrosis, and chronic inflammation. FAS-mediated apoptosis was enhanced contrary to p53-mediated apoptosis. Also noted that the upregulation of oncogenic proteins and the downregulation of tumor suppressor proteins. Red circle: maximum expression of upregulated proteins. Blue circle: minimum expression of downregulated proteins.

https://doi.org/10.1371/journal.pone.0261797.g016

PTX stimulated SHH/PTCH signaling by upregulating SHH and PTCH1, but reduced the expression of GLI1, and subsequently attenuated Notch/Jagged signaling by downregulating Notch1 and Jagged2. PTX-treated RAW 264.7 cells appeared to be differentiated into mature macrophages by upregulation of α-actin, VE-cadherin, CaM, TGase 2, PKC, p-PKC1α, AP1M1, FAK, AND AKAP13, and showed a characteristics of neuromuscular differentiation by upregulating NSE-γ, GFAP, MYH2, and desmin.

PTX was found to have anti-fibrosis effect on RAW 264.7 cells by downregulating FGF2, CTGF, collagen 3A1, laminin α5, and α1-antitrypsin. And PTX-treated cells showed a state of oncogenic stress by upregulating survivin, YAP1, CEA, and 14-3-3θ, and downregulating ATM, BRCA2, MBD4, and DMBT1.

PTX affected the expression of angiogenesis proteins in RAW 264.7 cells positively or negatively, that is, angiogenin, VEGF-A, vWF, and CD106 (VCAM-1) were downregulated by 10 μg/mL PTX, while HIF1α, CMG2, VEGF-C, VEGFR2, and p-VEGFR2 were upregulated. On the other hand, PTX consistently increased the expression of osteogenesis proteins, BMP2, BMPR1B, osterix, RUNX2, osteocalcin, osteonectin, versican, and ALP, but decreased RANKL expression (Fig 16) .

Comparison between 10 μg/mL and 300 μg/mL PTX application in RWA 264.7 cells Proliferation-related protein expression by 10 μg/mL or 300 μg/mL PTX. 300 μg/mL PTX reduced the expression of proliferation-related proteins in RAW 264.7 cells compared to 10 μg/mL PTX, that is, Ki-67 by 8.5% at 48 h, CDK4 by 9.9% at 12 h, cyclin D2 by 16.6% at 12 h, E2F1 by 14.8% at 24 h, cMyc by 6.4% at 12 h, MAX by 5.1% at 12 h, and MAD1 by 8.6% at 48 h, but increased the expression of p-Rb1 by 2.7% at 24 h. 10 μg/mL PTX stimulated the proliferation of RAW 264.7 cells by upregulating Ki-67, CDK4, cyclin D2, E2F1, cMyc, MAX, and downregulating p-Rb1, while 10 μg/mL PTX showed anti-proliferative effect on RAW 264.7 cells by downregulating Ki-67, cyclin D2, E2F1, and MAX compared to the untreated controls (Fig 17A and 17B) .

RAS/NFkB signaling protein expression by 10 μg/mL or 300 μg/mL PTX. 300 μg/mL PTX reduced the expression of RAS/NFkB signaling proteins in RAW 264.7 cells compared to 10 μg/mL PTX, that is, KRAS by 11.3% at 12 h, pAKT1/2/3 by 11.3% at 12 h, p-ERK1 by 14.7% at 24 h, NFkB by 15.4% at 24 h, p-p38 by 6.9% at 24 h, GADD153 by 10.8% at 48 h, and p-PKC1α by 5.8% at 24 h. However, the expression of pAKT1/2/3 and p-PKC1α by 300 μg/mL PTX were increased to 102% at 48h and 106.9% at 12 h, respectively, compared to those by 10 μg/mL PTX. 10 μg/mL PTX enhanced RAS/NFkB signaling by upregulating KRAS, pAKT1/2/3, p-ERK1, NFkB, p-p38, GADD153, and p-PKC1α compared to the untreated controls, while 300 μg/mL PTX attenuated RAS/NFkB signaling by downregulating pAKT1/2/3, p-ERK1, NFkB, p-p38, and GADD153 (Fig 17C and 17D ).

Inflammatory protein expression by 10 μg/mL or 300 μg/mL PTX. 300 μg/mL PTX markedly decreased the expression of inflammation-associated proteins in RAW 264.7 cells compared to 10 μg/mL PTX, that is, TNFα by 11% at 12 h, CD4 by 21.8% at 24 h, CD80 by 6.9% at 24 h, M-CSF by 15% at 48 h, CXCR4 by 7.1% at 24 h, MMP1 by 19.3% at 48 h, and lactoferrin by 15.7% at 24 h, but increased the expression of an inflammation suppressor TGF-β1 by 23.5% at 48 h. It was evident 10 μg/mL PTX stimulated the inflammatory reaction of RAW 264.7 cells by upregulating TNFα, IL-6, CD4, CD80, M-CSF, CXCR4,MMP1, lactoferrin, and downregulating TGF-β1 compared to the untreated controls, while 300 μg/mL PTX showed anti-inflammatory effect on cells by downregulating TNFα, IL-6, CD4, M-CSF, MMP1, lactoferrin, and upregulating TGF-β1 (Fig 17E and 17F) . osteogenic effect by downregulating RUNX2, BMP2, and osteocalcin compared to the untreated controls (Fig 18G and 18H ).

The pharmacological effect of PTX was frequently investigated in different cell types including RAW 264.7 cells [19] [20] [21] [22] and animals [50] [51] [52] [53] by using higher dose PTX, 100-500 μg/mL, rather than the therapeutic dose in human (about 10 μg/mL). It was also reported that the low dose PTX, 10, 25, 50 mg/kg, led to an increase in the expression of caspase 3 and TNFα in the rat hippocampus following lipopolysaccharide (LPS)-induced inflammation [54] . And the TNFα production by lipopolysaccharide (LPS)-stimulated human alveolar macrophages was significantly suppressed in the presence of PTX at concentration of 2 mM and 1 mM (278.3 μg/mL), but not at 0.5 mM, 0.1mM (27.8 μg/mL), and 0.01 mM, while production of IL-1β, IL-6, and GM-CSF remained unaffected. These data indicate PTX showed anti-inflammatory effect selectively depending on its concentration [55] .

In the present study, RAW 264.7 cells, which are originally murine monocytes, were explored for PTX-induced protein expression changes by administrating with two different doses, 10 μg/mL PTX similar to the therapeutic dose in human, and 300 μg/mL PTX which was frequently used in cell and animal experiments. First of all, the 10 μg/mL PTX-induced effect was compared with the 300 μg/mL PTX-induced effect. However, in clinical application, even the therapeutic dose PTX, about 10 μg/mL, produces diverse side effects including belching, bloating, stomach discomfort or upset, nausea, vomiting, indigestion, dizziness, flushing, angina, palpitations, hypersensitivity, itchiness, rash, hives, bleeding, hallucinations, arrhythmias, and aseptic meningitis [56, 57] . Therefore, it is thought that the low dose 10 μg/mL PTXinduced protein expression may be more informative to know the real pharmacological effect of PTX in human than the high dose 300 μg/mL PTX-induced protein expression in cell culture. Therefore, in the present study, 10 μg/mL PTX-induced effect on cells was more extensively investigated than 300 μg/mL PTX-induced effect.

In the global protein expression of RAW 264.7 cells by 10 μg/mL PTX, a competitive nonselective phosphodiesterase inhibitor which is known to raise intracellular cAMP and activate PKA, actually enhanced RAS signaling by upregulating AKAP13, pAKT1/2/3, PKC, p-PKC1α, KRAS, and HRAS, and subsequently activated histone/DNA demethylation and acetylation by upregulating KDMD4, HMGB1, and PCAF, and downregulating HDAC10, MBD4, DMAP1, DNMT1 and EZH2, and subsequently induced cellular proliferation by upregulating cMyc/ MAX/MAD network proteins, Wnt/β-catenin signaling objective protein TCF1, proliferation activating proteins Ki-67, PCNA, PLK4, cyclin D2, and cdc25A in this study. On the other hand, the expression of CDK inhibitors, p14, p15/16, and p21 were compensatory upregulated. The double IP-HPLC to assess the amount of protein complex containing two different target proteins showed the increase of cMyc-MAX heterodimer (Fig 8E and 8F ) and β-catenin-TCF1 complex (Fig 9G and 9H ) which led to cell cycle progression [58] , and the decrease of cMyc-MAD1 heterodimer and CDK4-p27 complex concomitantly. On the other hand, the double IP-HPLC also revealed the increase of E2F1-Rb1 and CDK4-p21 complexes (Fig 9C and 9D ), which mitigated cell cycle progression [59] .

Although the expression of cMyc and MAX were decreased by 10 μg/mL PTX at 12, 24, and 48 h, the expression of MAD1 was increased in western blot and IP-HPLC, and the double IP-HPLC showed dominant increase of cMyc-MAX heterodimer and decrease of cMyc-MAD heterodimer versus the untreated controls (Fig 8E and 8F) . Therefore, it is suggested that cMyc and MAX are competitively utilized to form cMyc-MAX heterodimer against cMyc-MAD heterodimer at 12, 24, and 48 h after 10 μg/mL PTX treatment, and then the unbound The 10 μg/mL PTX-treated RAW 264.7 cells showed crosstalk between activated RAS and NFkB signalings, and were progressed to cytodifferentiation by upregulating some growth factors and SHH/PTCH signaling. Particularly, it is evident that the 10 μg/mL PTX-treated RAW 264.7 cells have potentials of neuromuscular and osteoblastic differentiation, which are able to influence on objective adjacent cells [63] [64] [65] . The active neuromuscular and osteoblastic differentiations were also observed in ICC and western blot in this study. On the other hand, regarding the expression of osteogenesis proteins, 300 μg/mL PTX downregulated the osteoblastic differentiation proteins, BMP2, RUNX2, osterix, osteocalcin, osteopontin, and osteonectin, but upregulated the osteoclastic differentiation protein, RANKL, and compensatory increased the expression of OPG compared to 10 μg/mL PTX.

In the present study, 300 μg/mL PTX consistently decreased the expression of proliferation-, RAS/NFkB signaling-, inflammation-, and osteogenesis-related proteins but apoptosis proteins compared to 10 μg/mL PTX, therefore, it is thought that the high dose 300 μg/mL PTX may somehow disturb the protein expressions and give a harmful effect on RAW 264.7 cells, and resulted in the increase of FAS-mediated apoptosis, whereas the low dose 10 μg/mL PTX showed characteristic protein expression of competitive non-selective phosphodiesterase inhibitor, which may be helpful for the investigation of PTX pharmacological effect in human.

Contrary to the neuromuscular and osteoblastic differentiation in PTX-treated RAW 264.7 cells, 10 μg/mL PTX suppressed fibroblastic differentiation and attenuated collagen production by downregulating the fibrosis-inducing proteins, FGF1, FGF2, TGF-β1, CTGF, collagen 3A1, 4, and 5, laminin α5, integrin β1, α1-antitrypsin, and upregulating the fibrosis-inhibiting proteins, plasminogen, CMG2, integrin α2 and α5. On the other hand, 10 μg/mL PTX increased the expression of endothelin-1 having a key role of vascular homeostasis. In the global expression of 10 μg/mL PTX-treated RAW 264.7 cells, the anti-fibrotic protein expression is closely relevant to the reduction of chronic inflammation-associated M2 macrophage polarization by downregulating CD68, CD106, lysozyme, MMP9, and α1-antitrypsin, and the low level of ROS damage and ER stress after 10 μg/mL PTX treatment. Therefore, it is suggested that 10 μg/mL PTX inhibits M2 type macrophage polarization through RAS/NFkB/ TNFα signaling [66] , and reduces ER stress through eIF2α/eIF2AK3/GADD153/ATF6 signaling, which negatively regulates the growth, TGF-β1, -β2, -β3, FGF1, FGF2, and CTGF, and extracellular matrix-associated proteins, collagen-3A1, -4, -5A, laminin α5, integrin β1, plasminogen, PAI-1, α1-antitrypsin, and elafin, and eventually resulted in anti-fibrotic effect on RAW 264.7 cells.

10 μg/mL PTX reduced the expression of many angiogenic proteins, including angiogenin, VEGF-A, VEGF-D, vWF, FLT-4, LYVE-1, FGF-2, CD1056 (VCAM-1), MMP-2, MMP-10, PAI-1, CD54, and CD56, but upregulated some angiogenic proteins responsible for wound and damage, that is, HIF1α, VEGF-C, VEGFR2, CMG2, plasminogen, endothelin-1, and CD44. These results indicate 10 μg/mL PTX primarily inhibited angiogenesis but secondarily maintained de novo angiogenesis for wound healing [67] .

In addition, 10 μg/mL PTX was found to increase the potential of oncogenesis by upregulating the oncogenic proteins, CEA, 14-3-3θ, survivin, mucin 4, and YAP1, and downregulating the tumor suppressor proteins, P53, Rb1, BRCA1, BRCA2, NF1, ATM, maspin, and DMBT1. Nevertheless, 10 μg/mL PTX did not increase the expression of DNA repair enzymes, MBD4 and PARP-1, and exogenous stress responsible proteins, JNK1 and JAK2, but showed the overexpression of antioxidant proteins, SOD1 and GSTO1/2, and cell protection proteins, HSP70, sirtuin 6, and leptin, and resulted in the attenuation of ER stress. Therefore, it is suggested that 10 μg/mL PTX does not exert oncogenesis in RAW 264.7 cells, but maintains the cellular homeostasis, even though there appears slight elevation of some oncogenic protein expression.

The 10 μg/mL PTX-induced protein expression changes of different signaling pathways were summarized in a diagram of Fig 19. The panoramic protein signaling diagram illustrated main axes of protein signaling pathways in cells based on IP-HPLC data obtained in this study, therefore, it may indicate the real status of pharmacological effect of PTX in RAW 264.7 cells. We thought this PTX-induced protein expression of different signaling pathways should be corrected or added by further precise protein expression investigation using different cells and animals.

PTX as a non-selective phosphodiesterase inhibitor showed different biological effect on RWA 264.7 cells depending on the concentration of low dose 10 μg/mL or high dose 300 μg/mL PTX. 10 μg/mL PTX enhanced a central protein expression pathways, RAS/NFkB signaling, and subsequently induced proliferation, epigenetic activation, protection, survival, The main axis of cellular signaling, that is, proliferation, RAS signaling, NFkB signaling, protection, survival and aging, and inflammation were consistently activated by 10 μg/mL PTX, and subsequently followed by activation of epigenetic modification, protein translation, Wnt/β-catenin, cMyc/MAX/ MAD network, neuromuscular and osteoblastic differentiation, acute inflammation, innate immunity, cell-mediated immunity, and FASmediated apoptosis, while inactivation of p53/Rb/E2F signaling, ER stress, angiogenesis, fibrosis, and chronic inflammation. Sky blue letter: downregulated expression (under 80%), Blue letter: downregulated expression (80-95%), Green letter: minimal change expression (95-105%), Red letter: upregulated expression (105-120%), Flower pink letter: upregulated expression (over 120%).

https://doi.org/10.1371/journal.pone.0261797.g019 PLOS ONE neuromuscular and osteoblastic differentiation, and stimulated acute inflammation, innate immunity, and cell mediated immunity, while reduced chronic inflammation, ER stress, and fibrosis but reactively increased FAS-mediated apoptosis and oncogenic potential in RAW 264.7 cells. On the other hand, 300 μg/mL PTX was found to decrease RAS/NFkB signaling compared to 10 μg/mL PTX, and subsequently attenuated proliferation, epigenetic activation, inflammation, neuromuscular and osteoblastic differentiation but increased apoptosis and fibrosis. 

IRE1α, and ATF6α. Therefore, it is suggested that 10 μg/mL PTX-treated in RAW 264.7 cells are relatively safe under control of cellular protection and homeostasis. And more, the 10 μg/mL PTX-induced RAS signal by cAMP accumulation activated epigenetic modification through histone/DNA demethylation or acetylation, and subsequently elevated protein translation, which eventually positively influenced on cell proliferation, differentiation, protection and survival of RAW 264.7 cells. 10 μg/mL PTX-induced NFkB signal also influenced on the expression of inflammatory protein positively or negatively in RAW 264.7 cells. It was found some acute inflammatory proteins (TNFα, IL-1, IL-6, MCP1, CXCR4, granzyme B, and MMP1), innate immunity proteins (β-defensin 1, lactoferrin, versican, TLR 3 and 4), and cell-mediated immunity proteins (CD4, CD8, CD80, HLA-DR, perforin, and CTLA4) were upregulated, while some chronic inflammatory proteins including IL-12, CD68, CD106, lysozyme, cathepsin C and G, COX2, and α1-antitrypsin were downregulated. Therefore, it is suggested 10 μg/mL PTX-treated cells are tend to differentiate into M1 type macrophages, which are pro-inflammatory type and important for phagocytosis and secretion of pro-inflammatory cytokines and microbicidal molecules to defend against pathogens, such as bacteria, virus [60-62], etc. This activation of M1 type macrophage polarization after 10 μg/mL PTX treatment was correlated with the increase of FAS-mediated apoptosis contrary to p53-mediated apoptosis in RAW 264.7 cells. On the other hand, 300 μg/mL PTX consistently decreased the expression of inflammatory proteins

Continuous infusion treatment with pentoxifylline in patients with severe peripheral vascular occlusive disease

Trental 400 in the treatment of intermittent claudication: results of long-term, placebo-controlled administration

Involvement of cAMP/EPAC/Akt signaling in the antiproteolytic effects of pentoxifylline on skeletal muscles of diabetic rats

Pentoxifylline inhibits platelet-derived growth factor-stimulated cyclin D1 expression in mesangial cells by blocking Akt membrane translocation

Protective effects of pentoxifylline on acute liver injury induced by thioacetamide in rats

Pentoxifylline inhibits TLR-and inflammasome-mediated in vitro inflammatory cytokine production in human blood with greater efficacy and potency in newborns

Pentoxifylline, dexamethasone and azithromycin demonstrate distinct age-dependent and synergistic inhibition of TLR-and inflammasome-mediated cytokine production in human newborn and adult blood in vitro

Journal of Huazhong University of Science and Technology Medical sciences = Hua zhong ke ji da xue xue bao Yi xue Ying De wen ban = Huazhong keji daxue xuebao Yixue Yingdewen ban

Pentoxifylline Regulates Plasminogen Activator Inhibitor-1 Expression and Protein Kinase A Phosphorylation in Radiation-Induced Lung Fibrosis

Pentoxifylline and vitamin E for treatment or prevention of radiation-induced fibrosis in patients with breast cancer

Role of pentoxifylline and vitamin E in attenuation of radiation-induced fibrosis. The Annals of pharmacotherapy

Preventive Effect of Phosphodiesterase Inhibitor Pentoxifylline Against Medication-Related Osteonecrosis of the Jaw: An Animal Study

Antifibrotic effects of pentoxifylline improve the efficacy of gemcitabine in human pancreatic tumor xenografts. Cancer science

Enhanced anticancer efficacy of histone deacetyl inhibitor, suberoylanilide hydroxamic acid, in combination with a phosphodiesterase inhibitor, pentoxifylline, in human cancer cell lines and in-vivo tumor xenografts. Anti-cancer drugs

Potential Use of Pentoxifylline in Cancer Therapy. Current pharmaceutical biotechnology

Pentoxifylline-Induced Apoptosis in Chronic Lymphocytic Leukemia: New Insights into Molecular Mechanism. Mini reviews in medicinal chemistry

Pharmacokinetics of orally administered pentoxifylline in humans

Pharmacokinetics of orally administered pentoxifylline in humans

Effects of pentoxifylline, pentifylline and gamma-interferon on proliferation, differentiation, and matrix synthesis of human renal fibroblasts. Nephrology, dialysis, transplantation: official publication of the European Dialysis and Transplant Association-European Renal Association

Cytokine release from microglia: differential inhibition by pentoxifylline and dexamethasone. The Journal of infectious diseases

Pentoxifylline Can Reduce the Inflammation Caused by LPS after Inhibiting Autophagy in RAW264.7 Macrophage Cells

Cyclic nucleotides differentially regulate the synthesis of tumour necrosis factor-alpha and interleukin-1 beta by human mononuclear cells

High Performance Liquid Chromatography Analysis of Human Salivary Protein Complexes

IP-HPLC Analysis of Human Salivary Protein Complexes

Immunoprecipitation high performance liquid chromatographic analysis of healing process in chronic suppurative osteomyelitis of the jaw

Wound healing protein profiles in the postoperative exudate of bisphosphonate-related osteonecrosis of mandible

Differential protein expression in the secretory fluids of maxillary sinusitis and maxillary retention cyst

Changes in oncogenic protein levels in peri-implant oral malignancy: a case report. Maxillofacial plastic and reconstructive surgery

Two different protein expression profiles of oral squamous cell carcinoma analyzed by immunoprecipitation high-performance liquid chromatography. World journal of surgical oncology

In vivo protein expression changes in mouse livers treated with dialyzed coffee extract as determined by IP-HPLC. Maxillofacial plastic and reconstructive surgery

In vitro protein expression changes in RAW 264.7 cells and HUVECs treated with dialyzed coffee extract by immunoprecipitation high performance liquid chromatography

4-Hexylresorcinol-in duced angiogenesis potential in human endothelial cells. Maxillofacial plastic and reconstructive surgery

Effects of 4-Hexylresorcinol on Protein Expressions in RAW 264.7 Cells as Determined by Immunoprecipitation High Performance Liquid Chromatography

Preliminary study on hydrogen peroxide-induced cellular responses in RAW 264.7 cells as determined by IP-HPLC

Effects of 4-Hexylresorcinol on Craniofacial Growth in Rats

Evaluation of phenotypic and functional stability of RAW 264.7 cell line through serial passages

Hypusine, a polyamine-derived amino acid critical for eukaryotic translation

Comparative studies on tissue transglutaminase and factor XIII

Mechanism of action of guinea pig liver transglutaminase. VII. Chemical and stereochemical aspects of substrate binding and catalysis

Extensive protein expression changes induced by pamidronate in RAW 264.7 cells as determined by IP-HPLC

4-hexylresorcinol-induced protein expression changes in human umbilical cord vein endothelial cells as determined by immunoprecipitation high-performance liquid chromatography

Vascular endothelial growth factor-C accelerates diabetic wound healing. The American journal of pathology

Tracking angiogenesis induced by skin wounding and contact hypersensitivity using a Vegfr2-luciferase transgenic mouse

Converging physiological roles of the anthrax toxin receptors

Regulation of endothelin-1 synthesis by endothelin-converting enzyme-1 during wound healing. The Journal of biological chemistry

Brown tumor diagnosed three years after parathyroidectomy in a patient with nail-patella syndrome: A case report

The heat shock protein 90 inhibitor, 17-allylamino-17-demethoxygeldanamycin, enhances osteoclast formation and potentiates bone metastasis of a human breast cancer cell line. Cancer research

Versican is produced by Trif-and type I interferon-dependent signaling in macrophages and contributes to fine control of innate immunity in lungs. American journal of physiology Lung cellular and molecular physiology

NOS1 S-nitrosylates PTEN and inhibits autophagy in nasopharyngeal carcinoma cells

Pentoxifylline improves the survival of spermatogenic cells via oxidative stress suppression and upregulation of PI3K/AKT pathway in mouse model of testicular torsion-detorsion

Pentoxifylline decreases post-operative intraabdominal adhesion formation in an animal model

Pentoxifylline exerts anti-inflammatory effects on cerebral ischemia reperfusioninduced injury in a rat model via the p38 mitogen-activated protein kinase signaling pathway

Pentoxifylline Ameliorates Mechanical Hyperalgesia in a Rat Model of Chemotherapy-Induced Neuropathic Pain

The Effect of Pentoxifylline on Passive Avoidance Learning and Expression of Tumor Necrosis Factor-Alpha and Caspase-3 in the Rat Hippocampus Following Lipopolysaccharide-Induced Inflammation

The differential effect of pentoxifylline on cytokine production by alveolar macrophages and its clinical implications. Respiratory medicine

Systematic review of the use of pentoxifylline and tocopherol for the treatment of medication-related osteonecrosis of the jaw. Oral surgery, oral medicine, oral pathology and oral radiology

Direct induction of cyclin D2 by Myc contributes to cell cycle progression and sequestration of p27

p21 is a critical CDK2 regulator essential for proliferation control in Rb-deficient cells

A Drug with Antiviral and Anti-Inflammatory Effects to Be Considered in the Treatment of Coronavirus Disease 2019. Medical principles and practice: international journal of the Kuwait University

Cytokine Release (Storm) Syndrome" Caused by SARS-CoV-2? Current pharmaceutical design

Pentoxifylline and complicated COVID-19: A pathophysiologically based treatment proposal

Reversal of Osteopenia in Ovariectomized Rats by Pentoxifylline: Evidence of Osteogenic and Osteo-Angiogenic Roles of the Drug. Calcified tissue international

A phosphodiesterase inhibitor, pentoxifylline, enhances the bone morphogenetic protein-4 (BMP-4)-dependent differentiation of osteoprogenitor cells

Involvement of Rho and p38 MAPK in endothelin-1-induced expression of PGHS-2 mRNA in osteoblast-like cells

Pentoxifylline in the treatment of radiation-induced fibrosis

The Crucial Role of Vascularization and Lymphangiogenesis in Skin Reconstruction. European surgical research Europaische chirurgische Forschung Recherches chirurgicales europeennes

We express our gratitude to the late Professor Je Geun Chi who encouraged us to perform IP-HPLC, and to the late Dr. Soo Il Chung who taught us the biological usefulness of IP-HPLC.