Fig 2: Induction of necroptosis in co-cultures of cancer and non-cancerous cells(A) Detection of RIPK3 and β-actin in Hepa 1-6-hEpCAM and 3T6 cells by immunoblotting. Cell lines are shown at the top, the molecules detected are shown on the left.(B and C) Hepa 1-6-hEpCAM cells were infected with the viruses shown at the top at MOIs of 0.003 or 0.01 for 2 h, seeded together with the same number of the cells shown on the left (B) or below (C), and cultured for 2 days. (B) EGFP signals in syncytial plaques are visible. Scale bar, 10 mm. (C) Detection of phosphorylated MLKL in lysates of infected cells by immunoblotting. Molecules detected are shown on the left.

Fig 3: Characterizing the effect of Dox treatment on the expression of apoptosis and necroptosis-associated genes in the small intestine of neonatal 3xTg-iAPcIECmice. (A–C) RT-qPCR analysis of expression of molecular markers for apoptosis (Casp3 [A], Casp8 [B], and Casp9 [C]) in the ileum of Dox (20 mg/kg, s.c.)-treated 3xTg-iAPcIEC mouse pups (3xTg) and their littermate controls (Ctr). n = 6–7/group. Data represents 3 independent experiments and shows mean values ± standard deviation. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001 (vs. Control). Multiple Student t-test. (D) Immunoblotting analysis of expression and activation of markers for apoptosis (CASP3 and CASP8) and necroptosis (MLKL, RIP3, and RIP1) in the ileum of Dox-treated 3xTg-iAPcIEC neonatal pups and their littermate controls. Representative image of 3 independent experiments with 5 mice per group. C, Control. CL, Cleaved. (E–G) RT-qPCR analysis of expression of molecular markers for necroptosis (Mlkl [E], Rip3 [F], and Rip1 [G]) in the ileum of Dox (20 mg/kg, s.c.)-treated 3xTg-iAPcIEC mouse pups (3xTg) and their littermate controls (Ctr). n = 6–7/group. Data represents 3 independent experiments and shows mean values ± standard deviation. ns, Not significant. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001 (vs. Control). Multiple Student t-test.

Fig 4: Interaction of p62 with RIPK3 and pMLKL in the retina. (A) Immunoblot and its quantitative (histogram) analysis show increase of p62 in photodamaged retina. (B, C) Immunohistochemistry shows increase and colocalization of p62 with RIPK3 (B) and pMLKL (C) in the photoreceptor IS of photodamaged mice. (D) Interaction of p62 with RIPK1 and RIPK3 was analyzed by immunoprecipitation assay.

Fig 5: LPS-induced cardiac dysfunction could be improved by RIPK3 deletion (n = 6/group). (A–D) Cardiac function was measured via echocardiography. (E–J) Cardiomyocyte mechanical parameters were monitored using a VEVO 2,100 high-resolution imaging system. Cardiomyocytes were isolated from LPS-treated mice and melatonin-treated mice. Then, the resting cell length, peak shortening, time-to-90% re-lengthening (TR90), time-to- shortening (TPS), the maximal velocity of shortening (+dL/dt) and the maximal velocity of re-lengthening (-dL/dt) were determined. *p < 0.05 vs. WT group. #p < 0.05 vs. WT + LPS group.