Fig. 4: ISO induces TFEB- and TFE3-dependent autophagy.

A, B Human osteosarcoma U2OS cells stably expressing GFP-TFEB fusion protein were treated with torin 1 (300 nM) and isobacachalcone (ISO, 25 μM) for 6 h. Representative images are shown in (A) and TFEB translocation was assessed by measuring GFP intensities in the nuclei (B). Scale bar equals 10 μm. Data are means ± SD of quadruplicates (^***P < 0.001 vs. DMSO/Ctr, Student’s t test). C, D U2OS cells were treated with torin 1 (300 nM) and ISO (25 μM) for 6 h, and then, endogenous TFE3 translocation was assessed by immunostaining (C). Nuclear TFE3 intensities are depicted in (D). Scale bar equals 10 μm. Data are means ± SD of quadruplicates (^***P < 0.001 vs. DMSO/Ctr, Student’s t test). E–G U2OS cells were treated with ISO (25 μM) for 6 h or were left untreated. Cytoplasmic and nuclear fractions were assessed for nuclear translocation of the transcription factors TFEB and TFE3 in parallel samples by SDS–PAGE. GAPDH and H3 were used to ensure equal loading in the cytoplasmic and nuclear fractions, respectively. Band intensities of TFEB, TFE3, GAPDH, and H3 were assessed and their ratios (TFEB or TFE3/GAPDH, and TFEB or TFE3/H3) were calculated in (F, G). (^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. cytoplasmic DMSO; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. nuclear DMSO; Tukey’s multiple comparisons test). H–K U2OS cells stably expressing GFP-LC3 either wild-type (WT) or knockout for TFEB were treated with torin 1 (300 nM) or ISO (25 μM) for 16 h. LC3-II expression and TFEB deficiency were visualized by SDS–PAGE and immunoblot (J). Band intensities of LC3-II and β-actin (ACTB) were assessed, and their ratio (LC3-II/ACTB) was calculated in (K). Representative images are shown in (H), and GFP-LC3 dots were quantified as indicators of autophagy (I). Scale bar equals 10 μm. Data are means ± SD of quadruplicates (^***P < 0.001 vs. untreated control; ^#P < 0.05 vs. WT; Tukey’s multiple comparisons test). L–O U2OS cells stably expressing GFP-LC3 either WT or knockout for TFE3 were treated with torin 1 (300 nM) and ISO (25 μM) for 16 h. LC3-II expression and TFE3 deficiency were monitored by SDS–PAGE and immunoblot (N). Band intensities of LC3-II and ACTB were assessed, and their ratio (LC3-II/ACTB) was calculated in (O). Representative images are shown in (L), and GFP-LC3 dots were quantified (M). Scale bar equals 10 μm. Data are means ± SD of quadruplicates (^*P < 0.05, P < 0.001 vs. untreated control; ^#P < 0.05 vs. WT; Tukey’s multiple comparisons test). P–S U2OS cell stably expressing GFP-LC3 either wild-type or double knockout for TFEB and TFE3 cells were treated with torin 1 (300 nM) and ISO (25 μM) for 16 h. LC3-II expression and TFEB/TFE3 deficiency were checked in parallel samples by SDS–PAGE and immunoblot (R). Band intensities of LC3-II and ACTB were assessed, and the ratio (LC3-II/ACTB) was calculated (S). Representative images are shown in (P), and GFP-LC3 dots were quantified as indicators of autophagy (Q). Scale bar equals 10 μm. Data are means ± SD of quadruplicates (^*P < 0.05, ^***P < 0.001 vs. untreated control; ^#P < 0.05, ^##P < 0.01 vs. WT; Tukey’s multiple comparisons test).