Overexpression of OATL1 delayed autophagosomal maturation. (A) Overexpression of OATL1 resulted in an increase in the amount of LC3-II. MEF cells stably expressing T7-OATL1 or not expressing T7-OATL1 (control) were cultured under nutrient-rich (N), starved (S), or replenished (R) conditions, and their lysates were analyzed by immunoblotting with anti-T7 tag antibody (top), antiactin antibody (middle), and anti-LC3 antibody (bottom). (B and C) Wild-type MEF cells under the same conditions as in A were fixed and stained with anti-LC3 antibody or anti-Atg16L1 antibody. The mean numbers of LC3-positive (B) or Atg16L1-positive (C) dots per cell are shown. Error bars represent the means ± SEM of representative data (n ≥ 100) from two independent experiments. ***, P < 0.001; Student’s unpaired t test (compared with the control under the same conditions). (D) Overexpression of OATL1 increased the number of residual autophagosomes under replenished conditions. MEF cells stably expressing GFP-LC3 alone (control) or GFP-LC3 and T7-OATL1 (T7-OATL1) under the same conditions as in A were fixed and stained with anti-Atg16L1 antibody. GFP-LC3 and Atg16L1 are shown in the top and bottom, respectively. Bar, 10 µm. (E) EM analysis of the residual GFP-LC3–positive structures. MEF cells stably expressing GFP-LC3 and T7-OATL1 (T7-OATL1) or GFP-LC3 alone (control) were cultured under starved or replenished conditions and then fixed, and ultrathin cryosections were examined by immuno-EM. Under starved conditions, colloidal gold particles, indicating the presence of GFP-LC3, were detected in typical autophagosome-like structures (arrows) in both control cells and T7-OATL1–expressing cells. In T7-OATL1–expressing cells, numerous gold particles (some indicated by arrowheads) were detected in typical autophagosome-like structures (arrows) and occasionally on amphisome-like structures (asterisks), which contained multiple vesicles under replenished conditions. L, multilamellar lysosomal structure. Bars, 100 nm.