Fig. 6

BCRP3 is induced by certain proteotoxic stresses to enhance aggrephagy for protein quality control. A, B qRT-PCR analysis of BCRP3 levels in HeLa cells treated with 10 µM MG132 (A) or 4 µM As₂O₃ (B) for the indicated time points. Data were normalized to GAPDH and expressed as means ± SD, n = 3. P values are determined by one-way ANOVA with Tukey’s post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. C, D Control or BCRP3-deficient HeLa cells transfected with GFP-LC3 and RFP-p62 were treated with 10 µM MG132 for 8 h (C) or 4 µM As₂O₃ for 24 h (D) and analyzed by confocal microscopy (left). Arrowheads indicate the RFP-p62 puncta that are not colocalized with GFP-LC3. Bars, 10 μm. Pearson’s coefficients for the colocalization between GFP-LC3 and RFP-p62 signals were calculated by Image J and shown in the graphs (right). Data are means ± SD from three independent experiments and 10 cells per group per experiment were counted. P values are determined by one-way ANOVA with Tukey’s post hoc test, ***P < 0.001. E, G Control or BCRP3-deficient HeLa cells were treated with 10 µM MG132 for 8 h (E) or 4 µM As₂O₃ for 24 h (G), stained for p62 and ubiquitin, and analyzed by confocal microscopy (left). Bars, 10 μm. Area of colocalization between p62 and ubiquitin was calculated by Image J and shown in the graphs (right). Data are means ± SD from three independent experiments and 10 cells per group per experiment were counted. P values are determined by one-way ANOVA with Tukey’s post hoc test, **P < 0.01, ***P < 0.001. F, H Western blot analysis using control or BCRP3-deficient HeLa cells treated with 10 µM MG132 for 12 h (F) or 4 µM As₂O₃ for 24 h (H). Ubiquitinated protein levels were quantified and shown on the bottom