Fig. 3: RIPK3 kinase is active in the nucleus.

a Confocal images of single-optical sections of Hela cells transiently expressing kinase-dead KD-RIPK3. Scale bars: 10 µm. b Quantification of the percentage of nuclear GFP-RIPK3 D160N transiently expressed in HeLa cells treated with TBZ with or without LMB or control-treated. 14–20 transfected cells of a representative experiment of two independent experiments were analyzed. Plots indicate averages ± S.E.M. c Immunoblot of Flag-RIPK3 in nuclear and cytosolic fractions of TBZ or control treated HeLa cells expressing EV, RIPK3 or NLS-RIPK3. Immunoblots are representative of two independent experiments. Uncropped images of Western blots are shown in Supplementary Fig. 16. d Cell death profile of HeLa cells transiently expressing CFP-RIPK3 or CFP-NLS-RIPK3 pre-treated with NSA or control (con) followed by treatment with TBZ; SYTOX Red uptake in the CFP⁺ population was analyzed; n = 3. e Confocal images of single-optical sections of HeLa cells transiently transfected with MLKL-GFP. Left panel: con: control treated; LMB: treated with LMB; GppNHp: treated with GppNHp; LMB + GppNHp: treated with LMB and GppNHp. Scale bars: 10 µm. f Quantification of the percentage of nuclear MLKL-GFP transiently expressed in HeLa cells treated with control (con), LMB, GppNHp, or LMB + GppNHp. 13–24 transfected cells were analyzed of one or two independent experiments (n = 1 (GppNHp treated); n = 2 (con, LMB and GppNHp + LMB treated). Plots indicate averages ± S.E.M