NEDD4L down-regulation increases autophagy. (A) NEDD4L expression was down-regulated in HeLa by RNAi. Cells were nutrient-starved for the indicated time periods in the presence or not of Clq. Protein extracts were analyzed by WB for the expression of LC3, ACTIN, and NEDD4L. Densitometric analysis of LC3II+Clq-LC3II control over ACTIN band is shown. Data are expressed as the mean ± SEM of three independent experiments (n = 3). (B) HeLa cells were treated as in A. Protein extracts were analyzed by WB for the expression of p62 and ACTIN. (C and D) NEDD4L expression was down-regulated by RNAi as in A; cells were nutrient-starved for the indicated time periods and fixed and labeled with anti-ATG16L or anti-LC3 antibodies (red puncta) and visualized by confocal microscopy. Analysis of the number of both ATG16L and LC3 puncta occurrence per cell is shown in the graph. Data are expressed as the mean ± SEM (n = 3); representative images are shown. Bar, 20 µm (>50 cells analyzed per sample). In A, data were analyzed by one-way ANOVA followed by Tukey post hoc test. *, P < 0.05. In C and D, data were analyzed by two-way ANOVA followed by Bonferroni’s multiple comparison post test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.