Cxcr2

To investigate the effect of p17 on phosphorylation status of MLC, STAT1 and STAT3 total lysates were prepared by solubilization of cells in E1A lysis buffer (250 mM NaCl, 50 mM Hepes pH 7.0, 0.1% NP40, 50 mM EDTA) containing phosphatase and protease inhibitors. To investigate the effect of p17 on total protein expression of collagen-I, α-SMA, CXCR2, syndecan-2, endothelin-1 and tubulin, proteins were prepared for electrophoresis by boiling in 1X SDS Sample buffer (50 mM Tris HCl pH 6.8, 2.5% beta mercaptoethanol, 2% SDS, 10% glycerol). Following transfer to nitrocellulose membranes (Bio-Rad) proteins were detected with the following primary antibodies: procollagen type I (Santa Cruz - sc-8787), α-SMA (Santa Cruz – sc-53015), CXCR2 (Santa Cruz – sc-7304), syndecan-2 (Santa Cruz – sc-365624), endothelin-1 (Santa Cruz – sc-21625), phospho-MLC (thr18/ser19) (Santa Cruz – sc-12896), total MLC (Santa Cruz – sc-28329), α-tubulin (Sigma – T 6074), phosphoSTAT1(tyr701) (Cell Signaling - #9171), total STAT1 (Cell Signaling - #9172), phospho STAT3(tyr705) (Santa Cruz – sc-7993) and total STAT3 (Santa Cruz – sc-8019). Primary antibodies were detected with the horseradish peroxidase (HRP)-labeled secondary antibodies. Proteins were visualized by SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific Inc.) according to the manufacturer's instructions.

RPMI media 1640 was obtained from the Sigma-Aldrich Chemical (St. Louis, MO, USA), as were all other reagents unless specifically stated otherwise. Fetal bovine serum (FBS), penicillin/streptomycin, and trypsin/EDTA were purchased from the Lonza (Walkersville, MD, USA). IL-8 protein was provided by R&D systems (Minneapolis, MN, USA). Antibodies of α-smooth muscle actin (α-SMA), E-cadherin, fibronectin, vimentin, collagen I, membrane type 1-matrix metalloproteinase (MT1-MMP), tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, ZO-1, β-catenin and CXCR2 were purchased from the Santa Cruz Biotechnology (Dallas, TX, USA). Human N-cadherin antibody was supplied by Abcam (Cambridge, UK). Human collagen IV antibody was purchased from Bioss Antibodies (Woburn, MA, USA). Mouse monoclonal β-actin antibody was obtained from Sigma-Aldrich Chemical. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and goat anti-mouse IgG were purchased from Jackson Immuno-Research Laboratories (West Grove, PA, USA). Essential fatty acid free bovine serum albumin (BSA) and skim milk were supplied by Becton Dickinson Company (Sparks, MD, USA). 4′,6-Diamidino-2-phenylindole (DAPI) was obtained from Santa Cruz Biotechnology.
Aesculetin (Sigma-Aldrich Chemical) was dissolved in dimethyl sulfoxide (DMSO) for live culture with cells; a final culture concentration of DMSO was <0.5 %.

Hepatic stellate cells or KCs were washed three times with PBS, harvested, and lysed in co‐immunoprecipitation (co‐IP) buffer, as previously described.27 The total cell lysate (5 mg protein) was subjected to immunoprecipitation with the appropriate antibodies, as indicated, overnight at 4°C with gentle agitation, followed by incubation with protein A/G Plus‐agarose for 2 or 4 hour at 4°C. The immunocomplex was washed three times and then mixed with SDS sample buffer and boiled for 5 minutes. For western blotting, co‐precipitates or whole cell extracts were resolved by SDS‐PAGE and blotted on PVDF membranes (Millipore, Bedford, MA, USA). The membranes were immunoblotted with the indicated antibodies and developed using an ECL detection system (Applygen, Beijing, China). EGFR (phospho Y845) (1:1000); EGFR (1:1000); Erk1 (pT202/pY204) + Erk2 (pT185/pY187) (1:2000); ERK1 + ERK2 (1:2000); CXCR1 (1:2000); CXCR2 (1:2000); α‐SMA (1:2000); Smad3 (phospho S213) (1:1000); Smad3 (1:1000); BRD4 (1:2000); C‐MYC (1:2000); EZH2 (1:2000) and β‐actin (1:5000), were all purchased from Santa Cruz Biotechnology. The expression levels of proteins were determined using ImageJ software.