Fig. 1. MLKL deficiency impairs the orthotopic growth of HCC tumors.

a RIPK3 expression in patient samples across different types of cancer. Data were extracted from TCGA database. b Reconstitution of RIPK3 restores the necroptotic signaling in human HCC cells. HepG2 cells were transfected with the empty vector or pcDNA3.1-hRIPK3-Flag for 24 h and necroptotic signaling was stimulated by 50 μM Z-VAD-FMK followed by 50 ng/mL TNFα plus 20 μM Birinapant (T/B/Z). c MLKL expression in cancer tissues and adjacent normal tissues in two independent cohorts of HCC patients. d Survival analysis of MLKL high- and low-expression patients. The same set of data as in c were used for the analysis. e Reconstitution of RIPK3 restores the necroptotic signaling in murine HCC cells. Hepa 1–6 cells were treated and analyzed as described in b. f Growth curves of MLKL-KO and control murine HCC cells in vitro. Hepa 1–6-luc cells were infected with lentivirus-delivered sgMLKL or negative control (sgNC). KO efficiency was assessed by immunoblotting and cell growth was monitored by the live-cell analysis system IncuCyte (n = 3). g, h Orthotopic tumor growth of MLKL-KO and control HCC cells in a syngeneic model. Cells as described in f were orthotopically implanted in the liver of C57BL/6 mice (n = 13 for sgNC; n = 10 for sgMLKL #1 and sgMLKL #2). Tumor growth was monitored by whole-animal imaging. Endpoint tumor weight was shown in g. Representative luminescent images were shown in h. Data are represented as means ± SEM. Two-tailed Student’s t-test was used for statistical analysis. ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001.