Rhil 1α

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).

Human keratinocyte cell line, HaCaT cells, were obtained from China Center for Type Culture Collection (Wuhan, China). HaCaT cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) (both from Gibco, CA, USA) in 5% CO₂ environment at 37 °C. HaCaT cells were treated with 10 ng/mL recombinant human (rh) IL-17A (R&D, Minneapolis, USA), 10 ng/mL rh OSM (R&D), 10 ng/mL rh TNF-α (R&D), 10 ng/mL rh IL22 (R&D), and 10 ng/mL rh IL1-α (R&D) in combination for 6, 12, 24, or 48 h. Transfection was performed according to previously described methods (45). For GSDME knockdown, HaCaT cells were transfected with pGLVH1/GFP + Puro lentivirus vector containing NC shRNA (shNC sequence: 5’-TTCTCCGAACGTGTCACGT-3’) or GSDME shRNA (shGSDME sequence: 5’-GCAGAAGTGTGTGATCTCTGA-3’) (GenePharma, Shanghai, China). Stable knockdown HaCaT cells were obtained and selected by puromycin incubation.