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. 2022 Mar 16;221(5):e202112024. doi: 10.1083/jcb.202112024

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© 2022 Wang et al.

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Figure 5. — Ro-3306 promotes lysosomal function and biogenesis in a TFEB-dependent manner. (A) Western blotting assays showing the cytosolic and nuclear levels of endogenous TFEB. Treatment with 10 μM Ro-3306 or vanillic acid does not affect the levels, while 3-h Torin 1 treatment decreases the cytosolic level and increases the nuclear level. Phosphorylation of TFEB in whole-cell lysates, shown by the band shift, is affected by Torin 1, but not Ro-3306 or vanillic acid treatment. (B and C) In HeLa cells stably expressing TFEB-GFP, TFEB-GFP is predominantly localized in the cytoplasm (B). Treatment with 10 μM Ro-3306 (C) causes no change in the distribution of TFEB-GFP in HeLa cells. The nuclei of HeLa cells were stained by DAPI (blue channel) in B and C. (D) qRT-PCR assays showing upregulation of mRNA levels of lysosomal genes in HeLa cells treated with 20 μM Ro-3306 for 24 h. The level of the corresponding mRNA in control cells is set to 1.0. Data (normalized by GAPDH level) are shown as mean ± SEM (n = 3 for each bar). *, P < 0.05; **, P < 0.01; *** P < 0.001. (E–H) Compared with control cells, more lysosomal structures are labeled by anti-LAMP1 antibody (E) or stained by LysoTracker (F), DQ-BSA (G), and Magic Red (H) in HeLa cells treated with 10 μM Ro-3306. Lysosomes labeled by these markers are more scattered in Ro-3306–treated cells than in control cells. (I and J) In HeLa cells treated with 10 μM Ro-3306, the increased number of lysosomes stained by LysoTracker (shown in F) is suppressed by simultaneous depletion of TFEB (I) but is not affected by TFE3 depletion (J). (K) Quantification of the number of LAMP1, DQ-BSA, and Magic Red puncta in control cells and cells treated with 10 μM Ro-3306 or vanillic acid. Data are shown as mean ± SEM (n = 30, 30, 20, 30, 30, 30, 30, and 30 cells for the treatments shown on the x axis). ***, P < 0.001. (L) Quantification of the number of LysoTracker-stained puncta in control cells and cells treated with 10 μM Ro-3306 or Dinaciclib with or without knockdown of TFEB, TFE3, or CDK1. Data are shown as mean ± SEM (n = 30 cells for each bar). ***, P < 0.001. All measured values were statistically compared with the first dataset. Scale bars: 10 μm (B, C, and E–J). Source data are available for this figure: SourceDataF5.