Fig. 4

RIPK3 activation induces TRIM28 phosphorylation at serine 473. (A) HT-29 cells were treated with TSZ in a time-dependent manner. Cell lysates were analyzed by western blotting. (B) TSZ-treated HT-29 cells were stained with S824 and S473 phospho-TRIM28 antibodies and analyzed by confocal fluorescence microscopy (green: S824 or S473 phospho-TRIM28; blue: DAPI). (C) HT-29 cells were treated for 4 h with doxorubicin (2 μM), etoposide (100 μM), TSZ, TNF-α + CHX (5 μg/ml) + zVAD, or TRAIL (100 ng/ml) + SZ. (D) HT-29 cells were treated with TRAIL, TRAIL+ SZ, or TSZ for the indicated times, and cell lysates were analyzed by western blotting. (E) HT-29, SNU620, and ML-1 cells were treated with TSZ for the indicated times, and cell lysates were analyzed by western blotting. (F) HT-29 cells were pretreated with Nec-1 and GSK’872 for 1 h and with TSZ for 4 h. Cell lysates were analyzed by western blotting (left). Cells were also stained and visualized by confocal fluorescence microscopy (green: phospho-MLKL; red: S473 phospho-TRIM28; blue: DAPI) (right). (G) HT-29 cells stably expressing RIPK3 shRNA or the non-silencing control were treated with TSZ for the indicated times. (H) Cells from (G) were treated with TSZ for 4 h, stained, and visualized by confocal fluorescence (green: S473 phospho-TRIM28; blue: DAPI)