Fig. 4

TLR3 ligand, Poly(I:C) and Smac mimetic induce RIPK1-dependent apoptosis and necroptosis in CCA cell lines.a KKU100 cells were pretreated with 60 μM RIPK1 inhibitor (Nec-1, necrostatin-1) and 50 nM Smac mimetic for 2 h followed by transfection with 2.5 μg/ml Poly(I:C) (PS) or treatment with 10 ng/ml TNF-α (TS) (positive control) for 24 h. b KKU100 cells-expressing CRISPR control (CRISPR-V2) or CRISPR-RIPK1 (RIPK1) were pretreated with 50 nM Smac mimetic for 2 h followed by treatment as in (a) for 24 h. c KKU213 cells was treated as in (a) except for Smac mimetic was used at 5 nM and zVAD-fmk was also included. d KKU213 cells-expressing CRISPR control (CRISPR-V2) or CRISPR-RIPK1 (RIPK1) were treated as in (b). e HuCCT-1 cells were pretreated with 50 nM Smac mimetic and zVAD-fmk for 2 h, followed by transfection with 2.5 μg/ml Poly(I:C) (PSZ) or treated with 10 ng/ml TNF-α (TSZ) (positive control) for 24 h.f HuCCT-1 cells-expressing CRISPR control (CRISPR-V2) or CRISPR-RIPK1 (RIPK1) were treated as in (e) for 24 h. The representative knockout efficiency in (b) KKU100, (e) KKU213, and (f) HuCCT-1 cells were shown on right. Cell death was determined by Annexin V and PI staining and flow cytometry. Data from three independent experiments was presented as mean ± S.D.; * p < 0.05, **p < 0.01, *** p < 0.001