Cisplatin triggers the assembly of necrosome via autocrine production of TNFα in KYSE140 cells.
(A) KYSE140 cells were treated with 10 µM cisplatin for 6, 12, or 24 h, and the RIPK3 mRNA level was determined by RT-PCR. The relative density was compared to the GAPDH control. (B) The RIPK3 protein expression was determined by western blot analysis. The levels of RIPK3 protein were quantified and normalized to β-Actin. (C) Cisplatin promotes TNFα transcription. KYSE140 cells were treated with 10 µM cisplatin for the indicated time points, and the TNFα mRNA levels were analyzed by RT-PCR. The relative density was compared to the GAPDH control. (D) Cisplatin promotes autocrine production of TNFα. KYSE140 cells were treated with cisplatin for the indicated time points, and the level of TNFα secretion in the supernatant was measured by ELISA. (E) RIPK3 interacts with RIPK1 and MLKL. After treatment with cisplatin for 12 and 24 h, RIPK1 and MLKL were immunoprecipitated using anti-RIPK1 or anti-MLKL antibodies. RIPK1, RIPK3 and MLKL were detected by western blot analysis. (F) KYSE140 cells were treated with 10 µM cisplatin for 6 or 12 h. Mitochondrial and cytosolic fractions were isolated from the treated cells and analyzed for the indicated proteins with western blotting. AIF was used as a control for mitochondrial fraction and loading.