Figure 7.

TFEB activation improves lysosomal trafficking and corrects the localization of LAMP2A in cystinotic cells. A, WT and CTNS KO MEFs were treated with DMSO, genistein, or QX77 for 48 h, and RILP expression levels were analyzed by qPCR. Quantitation of RILP levels are from three independent experiments. Error bars represent S.E. *, p < 0.05, and **, p < 0.01, Student's t test. B, TFEB expression is down-regulated in cystinotic cells. Western blot analysis and quantification of the expression of endogenous TFEB in cystinotic cells are shown. Mean ± S.E. (n = 3). *, p < 0.05. C, exogenous expression of TFEB or TFEB-S3AR4A with constitutive nuclear localization (N) increases the perinuclear distribution of LAMP1- (upper panels) and LAMP2A (bottom panels)-positive organelles in cystinotic cells. Immunofluorescence of endogenous LAMP1 or LAMP2A (red) and the cellular distribution of GFP-TFEB or GFP-TFEB-S3AR4A in wild-type (WT) or Ctns^−/− cells was analyzed as described under “Materials and methods.” Scale bars, 20 μm. N, denotes nucleus. D, immunofluorescence analysis of endogenous LAMP1 and LAMP2A localization in Ctns^−/− cells either treated with genistein to activate TFEB or with vehicle (DMSO). Similar to that observed for TFEB overexpression, genistein treatment induces perinuclear localization of LAMP1 in cystinosis. Quantitative analysis indicates that genistein treatment improves the localization of LAMP2A at LAMP1-positive organelles. Mean ± S.E. (n = 3). ***, p < 0.001.