Fig. 1

Autophagic flux is upregulated in Cohen syndrome (CS) fibroblasts and CS iPSC-derived neurons. a Electron microscopic images of control and CS fibroblasts. b Quantitative analysis of EM images. The bar graph indicates the means ± SEM. *, p < 0.05 (Student’s t-test). c Western blot analysis showing autophagic flux in the presence or absence of a lysosomal inhibitor (bafilomycin A1 [BafA1], 100 nM) with or without rapamycin (100 nM, 4 h) using anti-LC3 or -GAPDH antibodies. d Relative expression of LC3-II normalized by GAPDH. The bar graph indicates the means ± SEM from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (one-way ANOVA followed by Tukey’s Multiple Comparison Test). e Electron microscopic images of synaptic regions in control and CS iPSC-derived neurons. f Western blot analysis showing autophagic flux in the presence or absence of a lysosomal inhibitor (chloroquine [CQ], 100 nM) using anti-LC3 or -β-actin antibodies. g Relative expression of LC3-II normalized by β-actin. The bar graph indicates the means ± SEM from three independent experiments. ***, p < 0.001 (one-way ANOVA followed by Tukey’s Multiple Comparison Test). h Western blot analysis showing autophagic flux in the presence or absence of a lysosomal inhibitor (BafA1, 100 nM) using anti-LC3 or -β-actin antibodies with WT or VPS13B KO HeLa cell (#1, or #2) lysates. i Quantification of band intensity of LC3-II or β-actin. The bar graph indicates the means ± SEM from three independent experiments. **, p < 0.01; ***, p < 0.001 (one-way ANOVA followed by Tukey’s Multiple Comparison Test). j The mRNA level of ATG4C determined by quantitative RT-PCR. The bar graph indicates the means ± SEM from three independent experiments. **, p < 0.01; ***, p < 0.001 (one-way ANOVA followed by Tukey’s Multiple Comparison Test). k Quantitative analysis of ATG4C expression in CS-derived neurons. (Inset) Western blot images of ATG4C expression in CS-derived neurons. The bar graph indicates the means ± SEM from five independent experiments. *, p < 0.05; **, p < 0.01 (one-way ANOVA followed by Tukey’s Multiple Comparison Test)