Tak 715

Peritoneal macrophages were prepared as described previously (Zhang et al. 2021b (link)) and were cultured in RPMI 1640 medium supplemented with 10% FBS (SH30071.03, HyClone, USA), 100 units/mL of penicillin, and 100 μg/mL of streptomycin. 2 μg/mL of LT [2 μg/mL of LF (172C, List Biological Labs) and 2 μg/mL of PA (171E, List Biological Labs)] was used to treat peritoneal macrophages for 3 h. HT-29 cells of indicated genotypes were cultured in DMEM supplemented with 10% FBS (10099-141, Gibco, USA), 100 units/mL of penicillin, and 100 μg/mL of streptomycin. 2 μg/mL of LT, 30 ng/mL of human TNF (PHC3011, Gibco), and 10 μmol/L of LCL-161 were used to induce cell death in HT-29 cells. Inhibitors used in this study were 2 μmol/L of SB202190 (S1077, Selleck, USA), 2 μmol/L of SB203580 (559389, Calbiochem), 2 μmol/L of TAK-715 (HY-10456, MedChemExpress), 2 μmol/L of U0126 (S1102, Selleck), 2 μmol/L of GDC-0994 (S7554, Selleck), and 10 μmol/L of SP600125 (S5567, Sigma–Aldrich, USA).

Primary human dermal fibroblasts were purchased from Life Technologies Co. (Carlsbad, CA, United States) and were cultured in medium 106 supplemented with low serum growth supplement (Life Technologies). Some cells were transfected with control (#AM4611) or Fn14 (#135142) siRNA (Life Technologies) (Xia et al., 2015 (link)). The transfection efficiency was verified by quantitative real-time PCR (qRT-PCR), which showed a >70% reduction in Fn14 mRNA expression (data not shown).
Prior to TWEAK stimulation, the cells were starved in 2% fetal bovine serum-supplemented medium for 24 h. The cells were stimulated with human recombinant TWEAK (0–250 ng/ml, 0–48 h; Cell Sciences, Canton, MA, United States). Some cells were pretreated with the specific inhibitors of the NF-κB (JSH-23, 20 μM), Wnt/β-catenin (XAV939, 20 nM), EGFR (erlotinib, 10 nM), p38 mitogen-activated protein kinase (MAPK) (TAK-715, 20 nM) and Smad3 (SIS3, 10 μM) signaling pathways (MedChemExpress, Monmouth Junction, NJ, United States) at 24 h before TWEAK stimulation.