S8605

Human hepatic stellate cell line LX‐2 was cultured in DMEM complete medium at 37 °C under 95% air and 5% CO₂. For hepatic stellate cell polarization in vitro, LX‐2 cells were treated with CRC‐derived CM, rhFGF19 proteins (50 ng mL⁻¹, R&D Systems) or rhIL‐1α (1 ng mL⁻¹, R&D Systems) for 24 h. Human FGF19 neutralizing antibody (10 µg mL⁻¹, AF969, R&D Systems), FGFR4 inhibitor fisogatinib (100 nм, S8503, Selleck) and BLU9931 (10 µм, A8706, APExBIO), JAK2 inhibitor fedratinib (10 µм, S2736, Selleck), STAT3 inhibitor C188‐9 (5 µg mL⁻¹, S8605, Selleck), and anakinra (20 mg mL⁻¹, Kineret) were used to inhibit iCAFs formation. WB, qRT‐PCR and ELISA were performed to detect the expression of myCAFs markers (α‐SMA, ACTG2, COL1A1, and COL2A1) and iCAFs markers (IL1A, IL1B, IL6, CXCL1, CXCL5), and the phosphorylation of JAK2‐STAT3 signaling pathway. Flow cytometry (FC) was performed to detect the expression of iCAF surface marker PDGFRα (1:250, 3174, Cell Signaling Technology) and myCAF marker α‐SMA (1:200, ab7817, Abcam). Data was collected and analyzed on a FACSCalibur flow cytometer (BD) FlowJo software (USA). Immunofluorescence (IF) staining was performed to detect the expression of pan‐CAF marker podoplanin (PDPN; 1:500, ab10288, Abcam) and iCAF marker IL‐6 (1:100, ab233706, Abcam). Images were acquired on a fluorescence microscope (Olympus).

RIPA lysis buffer was used to extract proteins from tissues or cell lines and protein samples (50 μg) were resolved by SDS-PAGE and then probed with indicated antibodies. CYB561D2, FAS ligand (FASLG), TGF-β2 (TGFB2), CD70, PD-L1, PD-L2, CCL2, TDO2, total STAT3, p-STAT3 (Tyr705) antibodies were from Sigma-Aldrich (SAB2500281), abcam (ab15285), abcam (ab36495), abcam (ab77868), abcam (ab205921), abcam (ab187662), abcam (ab186421), abcam (ab123403), CST (4904) and CST (9131), respectively. STAT3 inhibitor C188-9 was from selleckchem (S8605) [16 (link)]. The band density was analyzed with Image J and the density of target protein is normalized by Tubulin band. There are four biological replicates for each WB experiment.