Fig. 4.

Sec62 regulates PERK/ATF4 expression and activates autophagy accompanied by FIP200/Beclin-1/Atg5 upregulation. The WB assay demonstrated that Sec62 could affect PERK and ATF4 expression and showed no effect on IRE1, JNK, and ATF6 (A, B). Sec62 correlated with autophagy activation indicated by LC3 II, and P62 was identified via WB assays (C, D). Transmission electron microscopy revealed more autophagolysosomes (red arrows) or autophagosomes (blue arrows) in GC cells with higher Sec62 expression (E). Immunofluorescence assays after mRFP–GFP–LC3 double-labeled adenovirus transfection indicated increased LC3II expression (red or yellow dots) in GC cells with higher Sec62 levels (F, G). The CO-IP assay showed that exogenous FLAG-tagged Sec62 (the lentivirus bears a FLAG tag) is associated with endogenous LC3II (H). Immunofluorescence co-localisation assays also supported the relationship between Sec62 and LC3II. The Manders Overlap Coefficient (MOC) was 0.937 (I). Western blotting revealed a positive correlation between Sec62 and FIP200, Beclin-1, and Atg5 expression (J–K). *p < 0.05, **p < 0.01, ***p < 0.0001