Fig. 3: p27 affects autophagosome positioning in glucose-deprived cells.

A p27^+/+ and p27^−/− MEFs were glucose deprived for the indicated times and immunostained for LC3B. β-catenin was used to visualize cell shape and DNA was stained with Hoechst 33342. Scale bars are 50 µm. B Mean percentage of cells with over 50% of LC3B⁺ vesicles in the perinuclear region. n = 17 (p27^+/+) and n = 18 (p27^−/−) images from three experiments as described in (A) were used for quantification. Autophagosome distribution was evaluated based on a percentage of LC3 integrated density in perinuclear area (designed by region of interest [ROI]) versus LC3 integrated density in the whole cell. Graph show means ± SEM. C p27 immunoblot of p27^−/− MEFs retrovirally infected with p27 or empty vector. β-Actin was used as loading control. D LC3B immunostaining of MEFs infected with p27 or empty vector glucose deprived for 48 h. DNA was stained with Hoechst 33342. Scale bars are 50 µm. E Mean percentage of cells with over 50% LC3B⁺ vesicles in the perinuclear region from experiments as described in (D) from n = 3 independent experiments. Autophagosome distribution was evaluated as in (B). Graph show means ± SEM. B, E Statistical significance was evaluated by two-tailed Student’s t test with Welch’s correction.