Enhancing TFEB-Mediated Cellular Degradation Pathways by the mTORC1 Inhibitor Quercetin

Figure 3

Enhancing RPE cell degradation capacity by quercetin. (a) Measurement of the POS turnover rate. ARPE-19 cells were treated with 10 μM quercetin, or vehicle control, for 16 hr and subsequently were loaded with purified POS (5 : 1 ratio, POS : RPE) for 3 hr. After stringent washes, the rates of degradation of engulfed POS were measured by Western blot analyses of rhodopsin. To study the effects of lysosome inhibitors, cells were treated with 10 μM of E64d and pepstatin A or 10 μM CQ for 16 hr and then loaded with POS. (b) Effects of quercetin on LC3 lipidation. Cells were treated with the indicated concentration of quercetin, with or without 10 μM CQ, for 16 hr. Quantification data are presented in (c). (d, e) Western blot analyses of p62 protein in ARPE-19 cells treated with quercetin alone or with CQ. (f) Immunostaining of LC3 punctum formation after quercetin and CQ treatment; quantification data are presented in (g). (h, i) Effects of quercetin and vinblastine treatments on LC3 lipidation. Data presented are averages from 3–5 independent experiments (mean ± SEM). , , and . One-way ANOVA and Dunnett’s post hoc test.

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