FOXC2 ChIP-qPCR for HSC Activation

HSCs, cultured on 0.4 or 25.6 kPa hydrogels, were harvested for ChIP assay, ChIP assay was conducted using an EZ-Magna-ChIP HiSens kit (Millipore, #17-10461) as previously reported13 (link). Briefly, HSCs fixed with 1% formaldehyde and scraped from the hydrogels were subjected to nuclear lysis to release cross-linked protein/DNAs. The nuclear extract was then subjected to sonication and immunoprecipitation with anti-FOXC2 antibody (Abcam, #ab5060) or control rabbit IgG (Santa Cruz Technology, #sc-2027). Precipitated DNA fragments containing the promotor of ACTA2 (α-SMA), CTGF, FN1 and Col1A1 were quantitated by qPCR with the pair of primers. JASPAR software was used to predict the FOXC2 binding with the promoter of HSCs activation markers24 (link). The primers for ChIP-qPCR were displayed in Table S2.