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. 2021 Jun 14;9(6):e002722. doi: 10.1136/jitc-2021-002722

Figure 2.

Figure 2

TFEB and TFE3 mediate PPP-induced autophagy. (A–C) U2OS-GFP-LC3 wild-type (WT) or ATG5 knockout cells were treated with PPP (10 μM) or torin1 (300 nM) for 6 hours. Then the cells were fixed and GFP-LC3 dots were quantified. Scale bar equals 10 μm. Data are means ± SD of four replicates ***p<0.001 vs untreated control; ###P<0.001 vs WT; Tukey’s multiple comparisons test). (D, E) U2OS cells stably expressing GFP-TFEB fusion protein were treated with PPP (10 μM) for 16 hours and torin1 was used as positive control. nuclear GFP intensities were measured (E) and representative images are shown in D. Scale bar equals 10 µm. Data are means±SD of three replicates (**p<0.01, ***p<0.001 vs DMSO/Ctr, Student’s t-test). (F, G) U2OS cells were treated with PPP (10 μM) for 16 hours and torin1 was used as positive control and then endogenous TFE3 translocation was assessed by immunostaining (F) and TFE3 nuclear intensities are depicted (G). Scale bar equals 10 µm. Data are means±SD of three replicates (**p<0.01, ***p<0.001 vs DMSO/Ctr, Student’s t-test). (H) U2OS cells were treated with PPP (10 μM) for 16 hours or were left untreated. Cytoplasmic and nuclear fractions were separated and assessed for nuclear translocation of TFEB and TFE3 by SDS-PAGE. band intensities of TFEB, TFE3, GAPDH and H3 were assessed and their ratio (TFEB or TFE3/GAPDH, and TFEB or TFE3/H3) was calculated (online supplemental figure S3). (I–Q) U2OS-GFP-LC3 cells WT or single as well as double knockout for TFEB and TFE3 were treated with PPP (10 μM) or torin1 for 16 H. LC3 II expression and TFEB/TFE3 deficiency by knockout were checked by SDS-PAGE and parallel immunoblot (K, N, Q). Band intensities of LC3-II and β-actin (ACTB) were assessed, and their ratio (LC3-II/actin) was calculated (online supplemental figure S3). representative images are shown in (I, L, O), and GFP-LC3 dots were quantified as indicator of autophagy (J, M, P). Scale bar equals 10 μm. Data are means±SD of four replicates (***p<0.001 vs untreated control; #P<0.05, ##P<0.01, ###P<0.001 vs WT; Tukey’s multiple comparisons test). PPP, picropodophyllin SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.