OSW-1 triggers necroptosis in colorectal cancer cells through the RIPK1/RIPK3/MLKL signaling pathway facilitated by the RIPK1-p62/SQSTM1 complex

Figure 5 Impairment of the autophagic flux results in the accumulation of p62/SQSTM1, and p62/SQSTM1 can interact with RIPK1. A: The expression levels of p62/SQSTM1 and LC3II were evaluated in HT29 and HCT116 cells after exposure to OSW-1 for 24 h; B: The gene expression levels of p62/SQSTM1 and LC3 were assessed in HT29 and HCT116 cells after 24 h of exposure to OSW-1; C: Representative images of HT29 cells and HCT116 cells infected with adenovirus expressing GFP-mCherry-LC3. Cells not treated with OSW-1 served as the control. The images show total autophagosomes (yellow puncta) and functional autophagolysosomes (red puncta); D: Coimmunoprecipitation was conducted to assess the interaction between RIPK1 and p62/SQSTM1, and the results were analyzed through western blotting; E: Representative confocal microscopy images showing the colocalization of p62/SQSTM1 and RIPK1 in HT29 and HCT116 cells after exposure to OSW-1 for 24 h, with or without the addition of necrostatin-1. ^aP < 0.05 and ^bP < 0.01 vs Control. Each data point represents the mean ± SE. PI: Propidium iodide; Nec-1: Necrostatin-1.