Phosphorylated MLKL (pMLKL) was analyzed by Western blot analysis, and ОІ-actin and total MLKL served as loading control. (D) KKU213 and RMCCA-1 were pretreated with DMSO or 5 nM Smac mimetic and 20 ОјM zVAD-fmk for 2 h followed by treatment with 1 ОјM gemcitabine as indicated times. of three independent experiments are shown * p < 0.05, ** p < 0.01, *** p < 0.001. Cell death was determined by Annexin V and PI staining and flow cytometry. RIPK1 and RIPK3 knockout or MLKL knockdown (B) KKU213 and (C) RMCCA-1 cells were pretreated with DMSO or 5 nM Smac mimetic and 20 ОјM zVAD-fmk for 2 h followed by treatment with 1 ОјM gemcitabine for 72 h (KKU213) and 48 h (RMCCA-1). (A) MMNK1, KKU213, and RMCCA-1 were pretreated with DMSO or 5 nM Smac mimetic for 2 h in the presence of 20 ОјM zVAD-fmk followed by addition of 0.01, 0.1, 1, or 10 ОјM gemcitabine for 72 h (MMNK1 and KKU213) and 48 h (RMCCA-1). Smac mimetic and gemcitabine synergistically trigger necroptosis upon caspase inhibition. The representative knockdown efficiency was shown on right. (D) KKU213 and RMCCA-1 were infected with shRNA control (shNT) or shRNAs targeting two different sequences of MLKL (shMLKL1, shMLKL2). The representative knockout efficiency was shown on right. (C) KKU213 and RMCCA-1 were infected with CRISPR-V2, CRISPR-RIPK1, or CRISPR-RIPK3 to generate RIPK1 and RIPK3 knockout cells. HT-29 cells were included as a positive control for pMLKL (B) KKU213 and RMCCA-1 were pretreated with 60 ОјM necrostation-1 (Nec-1), 10 ОјM GSKвЂ™872 (GSK), or 1 ОјM necrosulfonamide (NSA) and Smac mimetic/zVAD-fmk for 2 h followed by TNF-О± treatment for 24 h. (A) MMNK1, KKU213, and RMCCA-1 were treated with TSZ for 8 h and 16 h, phosphorylated MLKL (pMLKL) was analyzed by Western blot analysis, and ОІ-actin and total MLKL served as loading control. TNF-О±/Smac mimetic-induced cell death upon caspase inhibition is required RIPK1, RIPK3, and MLKL proteins. Percentage of cell death (AnnexinV+/PI- and AnnexinV+/PI+) were determined by Annexin V and propidium iodide (PI) staining and flow cytometry. Smac mimetic and zVAD-fmk were pretreated for 2 h followed by treatment with TNF-О± for 24 h and 48 h. (D) RIPK3-expressing cells were treated as in C except KKU213 and RMCCA-1 were treated with 10 nM Smac mimetic and HuCCT-1 were treated with 25 nM Smac mimetic. (C) RIPK3 deficient cells, MMNK1, KKU100, and KKU214 were treated with 10 ng/ml TNF-О± (TNF-О±, complex I), TNF-О± and Smac mimetic, SM-164 (10 nM in MMNK1 and KKU214, or 25 nM in KKU100) (TS, complex IIa, apoptosis), or TNF-О± and Smac mimetic in the presence of 20 ОјM zVAD-fmk (TSZ, complex IIb, necroptosis) for 24 h and 48 h. (A) Key necroptotic proteins RIPK1, RIPK3 and MLKL (B) Protein expression of FADD and caspase-8 were analyzed in 7 different human CCA cell lines and a nontumor human cholangiocyte cell line, MMNK1 using Western blot analysis and ОІ-actin served as loading control. RIPK1, RIPK3 and MLKL-expressing CCA cell lines are sensitive to TNF-О±/Smac mimetic-induced cell death upon caspase inhibition. The cutoff of |Log2FC| was 1 and the cut-off * indicates p-value < 0.01. The red boxplot indicates tumor and the grey boxplot indicates normal tissue. RNA sequencing data were retrieved from The Caner Genome Atlas (TCGA) and were analyzed by an online tool GEPIA.

The mRNA expression of RIPK1, RIPK3, and MLKL was obtained from 36 CCA patients and 9 normal bile ducts. Different expression of key necroptotic proteins including RIPK1, RIPK3 and MLKL in CCA tissues.

Smac registration key