Fig. 2

Effects of RIPK3 inhibition on cell death in cardiomyocytes. Adult mouse cardiomyocytes isolated from RIPK3 knockout (RIPK3-KO) and wild-type mice (WT) were incubated with high glucose or mannitol (25 mmol/L) in the presence of GSK’872 and CHO alone or in combination for 24 h. a The levels of phosphorylated MLKL were determined by western blot analysis. Upper panel: a representative western blot for phosphorylated MLKL (p-MLKL), total MLKL and GAPDH; Bottom panel: quantification of p-MLKL relative to total MLKL. b LDH was measured in culture medium. c Necrotic cell death was assessed using PI staining. A representative micro-photograph for PI staining positive cells (red), nucleus Hoechst33342 staining (blue) and FITC-WGA staining for cell membrane (green). d Quantification of necrotic cell death. e and f LDH was measured in culture medium. g The phosphorylated levels of MLKL were determined in WT and RIPK3-KO cardiomyocytes. Upper panel: a representative western blot for p-MLKL, total MLKL and GAPDH; Bottom panel: quantification of p-MLKL relative to total MLKL. h LDH was measured in culture medium. Data are mean ± SD, n = 5 different cultures. *P < 0.05 versus Mannitol + Vehicle or Mannitol + WT and ^† P < 0.05 versus HG + Vehicle or HG + WT (ANOVA)