Fig. 7

Depletion of AGR2 resulted in aberrant translocation of 14-3-3ε or α-actinin 4 to the autophagosome. CMT-U27e, KO-S10, and KO-S4 seeded on coverslips were cultured in serum-free RPMI for 16 h, with or without adding 50 nM rapamycin (Rm). The cells were subsequently fixed and subjected to immunofluorescence staining of α-actinin 4 (A) or 14-3-3ε (B), together with LC3B. The resulting images were acquired using confocal microscopy. Puncta exhibiting LC3B (i.e., autophagosomes), α-actinin 4, or 14-3-3ε were identified using a Difference of Gaussian processing filter and presented in overlay images, denoted Merge (P). Scale bar, 20 µm. Colocalization of α-actinin 4 puncta with LC3B puncta, or 14-3-3ε puncta with LC3B puncta, were shown as overlap regions (yellow) in the enlargement, denoted Overlap (P). The number of LC3B puncta per cell (C) and the percentage of the LC3B puncta exhibiting colocalization with α-actinin 4 (D) or with 14-3-3ε (F) across all the experimental groups were quantified. Colocalization coefficients were measured for the puncta showing α-actinin 4-LC3B colocalization (E) and those showing 14-3-3ε-LC3B colocalization (G) per cell. The data in panels (C) through (G) were presented as the mean + SD