Figure 8.

UNC13D expression improves autophagy in a model of the lysosomal-storage disease cystinosis. (a–c) WT and ctns^−/- mouse embryonic fibroblasts were mock transfected (a), transfected with mCherry-UNC13D (b) or with mCherry-UNC13D-C2AC2B (C) expression vectors and immuno-stained with an anti-LC3B antibody. Representative confocal images are shown. Scale bar: 20 µm. (d) LC3B puncta density was calculated using Image Pro. Data are presented as mean ± SEM. At least 20 cells were analyzed. *p < 0.05. ANOVA followed by Fisher’s post-hoc test. (e–g) Analysis of the effect of UNC13D exogenous expression on autophagic flux in ctns^−/- cells under fed, starved and blocked conditions. ctns^−/- cells and ctns^−/- cells expressing GFP-UNC13D were fed, starved and/or treated with bafilomycin and analyzed for the expression of LC3B and SQSTM1 levels by Western blot. (e) Representative of 4 independent experiments. (f) and (g) The data are presented as Mean ± SEM. (h) and (i), Immunofluorescence analysis and quantification of the nuclear localization of TFE3 in wild-type, ctns^−/- and ctns^−/- cells expressing mCherry-UNC13D. Scale bar: 20 µm. n = 3; Mean ± SEM. At least 50 cells were analyzed. **p < 0.01 and ***p < 0.001.