Fig. 5.

Loss of CK1 abolishes RIPK3-S227 phosphorylation and blocks cell death. (A) Cell lysates from parental cells (HeLa:3xFlag-RIPK3) and KO cells were subjected to Western blotting with indicated antibodies. (B) Parental cells and KO cells were treated with DMSO or TSZ and cell viability was measured by the CellTiter-Glo assay. Viable cells are expressed as a percentage of DMSO-treated cells. Data are represented as mean ± SD of technical triplicates. (C) Parental cells or the CK1α/CK1ε double knockout (1α/1ε DKO) cells were treated with or without TSZ/NSA. Cell lysates were subjected to Western blotting with indicated antibodies. (D) Parental cells or the 1α/1ε DKO cells were treated with DMSO or TSZ and cell viability was measured by the CellTiter-Glo assay. (E) 1α/1ε DKO cells were transfected with siRNA against CK1δ or luciferase control for 72 h. The cells were then treated with TSZ/NSA for 4 h or 8 h and cell lysates were subjected to Western blotting. Antibody against phosphorylated MLKL-Ser358 is marked as p-MLKL. (F) The 1α/1ε DKO cells were transfected with siRNA against CK1δ or control for 72 h. The cells were then treated with DMSO or TSZ and cell viability was measured by the CellTiter-Glo assay. (G) HT-29 cells were transfected with two rounds of siRNA against control or a combination of siRNAs against CK1α, CK1δ, and CK1ε. The cells were then treated with or without TSZ/NSA and cell lysates were subjected to Western blotting with indicated antibodies. Antibody against phosphorylated S166 of RIPK1 is marked as p-RIPK1. (H) HT-29 cells were transfected with two rounds of siRNA as in G. The cells were then treated with DMSO or TSZ and cell viability was measured by the CellTiter-Glo assay.