Figure 2.

Increased oxidative stress causes autophagosome accumulation. (A) Representative western blots for p-MTOR, and MTOR with and without rapamycin (RAP) treatment. RAP samples were incubated with 200 nM RAP for 10 min, followed by 2 h without RAP before cell lysis. Quantification of p-MTOR:MTOR ratio. n = 10 for C₁; n = 7 for C₁+ RAP; n = 9 for KdVS₁; n = 6 for KdVS₁ + RAP; n = 10 for CRISPR₁; n = 7 for CRISPR₁+ RAP. Protein levels were normalized to loading control. Treatment efficiency was assessed by means of unpaired t test between untreated and RAP treated samples of C₁. (B) Representative western blots for SQSTM1 with and without RAP treatment. Quantification of SQSTM1. n = 8 for C₁; KdVS1; CRISPR1; n = 5 for C₁+ RAP; KdVS₁+ RAP; CRISPR₁+ RAP. Presented protein levels are relative to GAPDH. (C) Schematic representation of flux measurements through double-tagged mCherry-EGFP-LC3 transfection in iPSCs. (D) Example images of fluorescent LC3 detection after RAP and bafilomycin A₁ (BAF; 200 nM for 10 min prior to RAP) treatment. Quantification of autophagosomes. n = 23 for C₁+ RAP; n = 14 for C₁+ RAP+BAF; n = 18 for KdVS₁+ RAP; n = 14 for KdVS₁+ RAP+BAF; n = 22 for CRISPR₁+ RAP; n = 12 for CRISPR₁+ RAP+BAF. (E) Representative western blots for SOD1 and SOD1 protein quantification. Protein levels were normalized to GAPDH. n = 6 for C₁; KdVS₁; CRISPR₁. Statistical significance was tested by means of ordinary one-way ANOVA and Sidak’s multiple comparison test. (F) Schematic presentation and example western blots of SOD1 overexpression in KdVS₁ and CRISPR₁ iPSCs. (G) Representative images and quantification of SQSTM1 particles in iPSCs with and without SOD1 overexpression. n = 14 for C₁; n = 15 for KdVS₁; n = 15 for CRISPR₁. (H) Representative images of iPSC colonies stained with CellROX and fluorescence quantification. iPSCs were either untreated or treated overnight with 100 µM apocynin (APO). n = 26 for C₁; n = 23 for C₁+ APO; n = 12 for KdVS₁; n = 10 for KdVS₁+ APO; n = 26 for CRISPR₁; n = 28 for CRISPR₁+ APO. Fluorescence levels were normalized to the untreated control. Scale bar: 20 µm. (I) Representative images of iPSC colonies stained for SQSTM1 and particle analysis. iPSCs were either untreated or treated overnight with 100 µM APO. n = 27 for C₁; n = 24 for C₁+ APO; n = 33 for KdVS₁; n = 22 for KdVS₁+ APO; n = 23 for CRISPR₁; n = 23 for CRISPR₁+ APO. Fluorescence levels were normalized to untreated control. Scale bar: 20 µm. Data presented in this figure were collected in at least 3 independent experiments. Statistically significant differences were determined by means of Kruskal Wallis and Dunn’s multiple comparison test, if not mentioned differently. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001.