Fig. 7

Macrophage Notch1 deficiency-mediated TRAF6 enhances stress-induced hepatocyte necroptosis via modulating RIPK3 signaling activation. BMMs were isolated from Notch1^M−KO mice and transfected with the p-CRISPR-TRAF6 KO or control vector followed by LPS stimulation. A The schematic figure for macrophage/hepatocyte co-culture system. B ELISA analysis of supernatant TNF-α levels in LPS-stimulated BMMs (n = 3–4 samples/group). C LDH release from hepatocytes in co-cultures. Quantification of average cytotoxicity (% cell death) in different groups were plotted. (n = 3–4 samples/group). D BMMs transfected with the p-CRISPR-TRAF6 KO were stimulated with LPS, and then co-cultured with primary hepatocytes supplemented with or without H₂O₂ for 24 h. Western-assisted analysis of p-MLKL and RIPK3 in hepatocytes after co-culture. Representative of three experiments. E Immunofluorescence staining of TUNEL⁺ hepatocytes after co-culture (n = 4–6 mice/group). Scale bars, 40 μm. All data represent the mean ± SD. **p < 0.01