H3k4me2

Rat anti-mouse F4/80 antibody was purchased from Serotec; Rat anti-mouse CD31 was purchased from Dianova (Hamburg, Germany). Anti-actin and anti-smooth muscle actin (SMA) monoclonal antibodies were purchased from Sigma (St. Louis, MO). Antibodies directed against phopsho-AMPK alpha, phospho-p38 and phospho-JNK, histone H3 and H3K4-di (H3K4me2) and -tri (H3K4Me3) methylation, H3K27 tri-methylation and acetylated H2BK5 were purchased from Cell Signaling Technology (Beverly, MA). Anti-WDR5, anti-BiP, and anti-Ly6g antibodies were purchased from Abcam (Cambridge, UK). The selective inhibitors of LSD1 (GSK-2879552), WDR5 (OICR-9429), MKL1 (CCG-203971), CXCR2 (SB 225002), p38 (SB208530), and JNK (sp600125) were purchased from ApexBio (Boston, MA) and were dissolved in DMSO as stock solutions. DMSO (Sigma D2438) was added to the cell culture medium as control. The heparanase inhibitors H1001 and PG545 were kindly provided by HepaRx Ltd (Ness-Ziona, Israel) and Zucero Therapeutics (Darra, Queensland, Australia), respectively. Mouse CXCL2/MIP-2 Quantikine ELISA kit was purchased from R&D Systems (Minneapolis, MN). The MyD88 peptide inhibitory set was purchased from Novus Biologicals (Centennial, CO). Latent heparanase was purified from medium conditioned by CHO cells overexpressing heparanase essentially as described (25 ) and was added to cell cultures at 1 μg/ml.

Immunoblotting analysis was performed as previously described^{65 (link)}. Cells were washed twice with ice cold PBS, and harvested in protein lysis buffer (50 mM HEPES [pH7.4], 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mM glycerophosphate, 2.5 mM sodium pyrophosphate, 1 mM Na3VO4, 20 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 mM DTT, 1× complete protease inhibitor tablet [Roche]). Total cell lysates were separated on 4-12% Bis-Tris gels (Life Technologies), transferred onto PVDF membrane (Merck Millipore). The blots were probed with antibodies for H3, H3K4me2, H3K9me2, H3K27me2 (#9847 from Cell Signaling Technologies for above antibodies; 1:1000), H3K27me3 (Cell Signaling Technologies; #9733, 1:1000), SUZ12 (Cell Signaling Technologies; #3737, 1:1000), Ezh2 (Cell Signaling Technologies; #5246, 1:1000), RpAb46/48 (Santa Cruz Biotechnology; #33170, 1:1000), EED (Santa Cruz Biotechnology; #28701, 1:1000), p-S6K (Thr389, Cell Signaling Technologies; #9234, 1:1000) and S6K (Cell Signaling Technologies, MA, USA; #9202, 1:1000) as indicated in figures. Protein signals were detected using HRP conjugated secondary antibodies and enhanced chemiluminescence (ECL) western blotting detection regents (Thermo Fisher Scientific, MA, USA).