Fig. 3

Resibufogenin induces RIP3-dependent necroptosis through activating PYGL, GLUL and GLUD1. a Immunoblot analysis of necrotic proteins were determined after 24 h of resibufogenin treatment. Level of Bax, P53, and PGAM5 in cytosol and mitochondrial fractions from HCT116 cells treated with resibufogenin at the indicated concentrations for 24 h were evaluated by western blot analysis. β-Actin was used as a loading control. See also Additional file 1 and Additional file 2: Figures S7, S8. The gray value of each stripe has been calculated using quantity one software. b Confocal immunofluorescence of RIP3 (red, anti-RIP3) in the HCT116 and SW620 cells that were loaded with MitoTracker and DAPI (n = 3). ×1000 for all, scale bar = 1 μm. c RIP3^+/+and RIP3^−/− cells were treated with resibufogenin for 24 h. cells viability was measured by MTT assay (n = 6). See also Additional file 1 and Additional file 2: Figure S9. d The activity of three energy metabolism enzymes in the two cell lines (n = 3). e The protein expression of three energy—metabolizing enzymes in two cell lines. Data represent mean ± SEM, ^#P < 0.05, *P < 0.01, as determined by one-way ANOVA followed by Tukey’s multiple comparison test