Fig. 3

TLR3 ligand, Poly(I:C) and Smac mimetic trigger necroptosis upon caspase inhibition in CCA cell lines. a RIPK3-expressing cells, KKU213 and HuCCT-1 were pretreated with 20 μM zVAD-fmk and Smac mimetic (5 nM KKU213 and 50 nM HuCCT-1) for 2 h. The cells were transfected with 2.5 μg/ml Poly(I:C) for 24 h and 48 h. TNF-α/zVAD-fmk/Smac mimetic (TSZ) were represented as a positive control. KKU213 (left) and HuCCT-1 (right) cells were pretreated with 10 μM RIPK3 inhibitor (GSK’872) (b) or 1 μM MLKL inhibitor (NSA) (c) for 2 h. At the same time, the cells were pretreated with zVAD-fmk and Smac mimetic (SZ). After that the cells were treated as in (a). d KKU213 and HuCCT-1 cells-expressing CRISPR control (CRISPR-V2) or CRISPR-RIPK3 (RIPK3) were treated as in (a) for 24 h. The representative knockout efficiency was shown on right. e KKU213 and HuCCT-1 cells-expressing shRNA control (shNT) or shRNAs targeting two different sequences of MLKL (shMLKL1, shMLKL2) were treated as in (a). The representative knockdown efficiency was 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