Figure 6.
Necroptosis induced by TNF destabilizes SNARE complexes and cleaves STX17 to block LC3 degradation. (A) L929 cells stably expressing GFP-SNAP29 were exposed to 10 ng/ml TNF and 30 µM QVD (TQ) for indicated times. Then, cells were lysed and cleared cellular lysates were subjected to immunopurification using anti-GFP beads. Purified proteins were subjected to SDS-PAGE and analyzed by immunoblotting for indicated proteins. (B) L929 cells stably expressing GFP-SNAP29 were left untreated (medium, M) or exposed to 10 ng/ml TNF + 30 µM QVD (TQ), TQ plus 5 µM GSK’872 (TQG), or TQ plus 5 µM necrostatin-1 (TQN) for 4 h. Then, cells were lysed and cleared cellular lysates were subjected to immunopurification using anti-GFP beads. Purified proteins were subjected to SDS-PAGE and analyzed by immunoblotting for indicated proteins. (C) L929 cells were left untreated (medium, M) or exposed to 30 µM QVD (Q), 20 nM bafilomycin A1 (B), 10 ng/ml TNF (T), 10 ng/ml TNF + 30 µM QVD with or without 20 nM bafilomycin A1 (TQ or TQB) for indicated times. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and immunoblotting for STX17 and ACTB. (D) L929 WT, ripk3 KO or mlkl KO cells were left untreated or exposed to 10 ng/ml TNF + 30 µM QVD (TQ) for 4 h. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and immunoblotting for STX17, RIPK3, MLKL, and GAPDH. (E) L929 cells were transfected with non-targeting (siCtrl) or Prkaa1/Prkaa2 siRNAs (siPrkaa1/siPrkaa2). 48 h post transfection, cells were left untreated (medium, M) or exposed to 10 ng/ml TNF + 30 µM QVD (TQ) with or without 5 µM GSK’872 (G) for 4 h. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and analyzed by immunoblotting for indicated proteins. The density of each protein band was divided by the average of the density of all bands from the same protein on the membrane. Fold changes were calculated by dividing each normalized ratio (protein to loading control) by the average of the ratios of the control lane (siCtrl, medium). Statistical graphics represents mean + SD (n = 3). Statistical analysis was done by ordinary two-way ANOVA (corrected by Tukey’s multiple comparisons test). Statistically significant differences are only indicated for TQ and TQG (scr vs. Prkaa1/Prkaa2 siRNA) and for TQ vs. TQG (within each treatment). Statistically significant differences to control (siCtrl, medium) are depicted as letters directly above the bars. ** or b: P < 0.01, *** or c: P < 0.001, ns: non-significant. (F) L929 cells were left untransfected or transiently transfected with cDNA encoding 3xFLAG-MmRIPK3 for 24 h. Then untransfected cells were left untreated or exposed to 10 ng/ml TNF + 30 µM QVD (TQ) for 3 h. Cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and immunoblotting for indicated proteins