Figure S2.
Autophagy is required for SOD1 delivery to lysosomes. (A) Volcano plot of mCherry/GFP fluorescence intensity ratios in 293T cells transduced with the h2 gRNA sublibrary of the human CRISPRi-v2 library. The cells were stably expressing mCherry-GFP-SOD1(G93A) and dCas9. gRNAs targeting genes encoding components of the autophagy pathway were the top hits in the screen (highlighted in magenta). All other targeting sgRNAs are indicated in gray. (B) KEGG pathway analysis identifies autophagy as a major factor responsible for SOD1(G93A) transport into lysosomes. High-confidence factors were defined as having opposite phenotypes in the enhanced and inhibited sort gates and gene levels P < 0.01. Pathways with Benjamini–Hochberg false discovery rate (FDR) < 0.1 are shown. (C) Immunoblotting with anti-FIP200 and anti-ACTB antibodies in WT and FIP200 KO 293T cells. (D) Confocal images of VMP1 KO COS7 cells expressing mCherry-GFP-SOD1 under control (Ctrl) or starvation (Strv) conditions. Bars: 5 μm; insets, 2 μm. (E) In a GFP-Trap assay, endogenous LC3B is immunoprecipitated by GFP-TP53INP1. (F) Relative TP53INP1 transcription levels in NC and siTP53INP1 cells. Quantitative data normalized by ACTB levels are presented as mean ± SEM (n = 3). (G and H) Confocal images show that compared with control 293T cells, the number of LC3B puncta is reduced in siTP53INP1 cells upon starvation (Strv) (G). Quantification of LC3B puncta is presented as mean ± SEM (NC, n = 20; siTP53INP1, n = 20) (H). ****, P < 0.0001. Bars: 5 μm. (I) Immunoblotting shows that compared with control 293T cells, the levels of mCherry-SOD1, mCherry-SOD1(G93A), mCherry-SOD1(A4V), and mCherry-SOD1(G85R) remain unchanged under starvation (Strv) or starvation plus Baf treatment. (J) Immunoblotting shows that the Flag-SOD1 level is reduced following starvation plus cycloheximide (CHX) treatment for 24 h, and this reduction is slightly reversed by the addition of Baf. Quantification of SOD1 levels under different conditions, normalized by ACTB levels, is shown. (K) Immunoblotting verifies the KD efficiency of siSOD1 in 293T cells. (L) Compared with WT SOD1, the enzyme activities at pH 7.2 of SOD1(G93A), SOD1(A4V), and SOD1(G93A) are significantly reduced. Quantification of SOD activity is presented as mean ± SEM (n = 3). ****, P < 0.0001. (M) Compared with WT SOD1, the enzyme activities at pH 5.2 of SOD1(G93A), SOD1(A4V), and SOD1(G93A) are significantly reduced. Quantification of SOD activity is presented as mean ± SEM (n = 3). ****, P < 0.0001. (N) Compared with NC 293T cells under normal conditions, the lysosomal superoxide levels detected by HKSOX-2L probe are increased after starvation (Strv) and further elevated by siSOD1. Quantification of HKSOX-2L intensity is presented as mean ± SEM (NC Ctrl, n = 20; NC Strv, n = 20; siSOD1 Ctrl, n = 21; siSOD1 Strv, n = 21). ****, P < 0.0001. (O) Immunoblotting shows that levels of SOD1 are not changed in siTP53INP1 293T cells. (P and Q) Confocal images show that there is no difference in the lysosomal superoxide levels between control 293T cells and siTP53INP1 cells in the absence of starvation (P). Quantification of HKSOX-2L intensity is presented as mean ± SEM (NC, n = 20; siTP53INP1, n = 20) (Q). Bars: 5 μm. (R and S) Confocal images show that there is no difference in the lysosomal superoxide levels detected by HKSOX-2L between WT 293T cells and FIP200 KO cells under control (Ctrl) conditions. After starvation (Strv), the lysosomal superoxide levels are increased in FIP200 KO cells compared with WT cells (R). Quantification of HKSOX-2L intensity is presented as mean ± SEM (WT Ctrl, n = 20; WT Strv, n = 20; FIP200 KO Ctrl, n = 20, FIP200 KO Strv, n = 20) (S). ****, P < 0.0001. Bars: 5 μm. Source data are available for this figure: SourceData FS2.

Autophagy is required for SOD1 delivery to lysosomes. (A) Volcano plot of mCherry/GFP fluorescence intensity ratios in 293T cells transduced with the h2 gRNA sublibrary of the human CRISPRi-v2 library. The cells were stably expressing mCherry-GFP-SOD1(G93A) and dCas9. gRNAs targeting genes encoding components of the autophagy pathway were the top hits in the screen (highlighted in magenta). All other targeting sgRNAs are indicated in gray. (B) KEGG pathway analysis identifies autophagy as a major factor responsible for SOD1(G93A) transport into lysosomes. High-confidence factors were defined as having opposite phenotypes in the enhanced and inhibited sort gates and gene levels P < 0.01. Pathways with Benjamini–Hochberg false discovery rate (FDR) < 0.1 are shown. (C) Immunoblotting with anti-FIP200 and anti-ACTB antibodies in WT and FIP200 KO 293T cells. (D) Confocal images of VMP1 KO COS7 cells expressing mCherry-GFP-SOD1 under control (Ctrl) or starvation (Strv) conditions. Bars: 5 μm; insets, 2 μm. (E) In a GFP-Trap assay, endogenous LC3B is immunoprecipitated by GFP-TP53INP1. (F) Relative TP53INP1 transcription levels in NC and siTP53INP1 cells. Quantitative data normalized by ACTB levels are presented as mean ± SEM (n = 3). (G and H) Confocal images show that compared with control 293T cells, the number of LC3B puncta is reduced in siTP53INP1 cells upon starvation (Strv) (G). Quantification of LC3B puncta is presented as mean ± SEM (NC, n = 20; siTP53INP1, n = 20) (H). ****, P < 0.0001. Bars: 5 μm. (I) Immunoblotting shows that compared with control 293T cells, the levels of mCherry-SOD1, mCherry-SOD1(G93A), mCherry-SOD1(A4V), and mCherry-SOD1(G85R) remain unchanged under starvation (Strv) or starvation plus Baf treatment. (J) Immunoblotting shows that the Flag-SOD1 level is reduced following starvation plus cycloheximide (CHX) treatment for 24 h, and this reduction is slightly reversed by the addition of Baf. Quantification of SOD1 levels under different conditions, normalized by ACTB levels, is shown. (K) Immunoblotting verifies the KD efficiency of siSOD1 in 293T cells. (L) Compared with WT SOD1, the enzyme activities at pH 7.2 of SOD1(G93A), SOD1(A4V), and SOD1(G93A) are significantly reduced. Quantification of SOD activity is presented as mean ± SEM (n = 3). ****, P < 0.0001. (M) Compared with WT SOD1, the enzyme activities at pH 5.2 of SOD1(G93A), SOD1(A4V), and SOD1(G93A) are significantly reduced. Quantification of SOD activity is presented as mean ± SEM (n = 3). ****, P < 0.0001. (N) Compared with NC 293T cells under normal conditions, the lysosomal superoxide levels detected by HKSOX-2L probe are increased after starvation (Strv) and further elevated by siSOD1. Quantification of HKSOX-2L intensity is presented as mean ± SEM (NC Ctrl, n = 20; NC Strv, n = 20; siSOD1 Ctrl, n = 21; siSOD1 Strv, n = 21). ****, P < 0.0001. (O) Immunoblotting shows that levels of SOD1 are not changed in siTP53INP1 293T cells. (P and Q) Confocal images show that there is no difference in the lysosomal superoxide levels between control 293T cells and siTP53INP1 cells in the absence of starvation (P). Quantification of HKSOX-2L intensity is presented as mean ± SEM (NC, n = 20; siTP53INP1, n = 20) (Q). Bars: 5 μm. (R and S) Confocal images show that there is no difference in the lysosomal superoxide levels detected by HKSOX-2L between WT 293T cells and FIP200 KO cells under control (Ctrl) conditions. After starvation (Strv), the lysosomal superoxide levels are increased in FIP200 KO cells compared with WT cells (R). Quantification of HKSOX-2L intensity is presented as mean ± SEM (WT Ctrl, n = 20; WT Strv, n = 20; FIP200 KO Ctrl, n = 20, FIP200 KO Strv, n = 20) (S). ****, P < 0.0001. Bars: 5 μm. Source data are available for this figure: SourceData FS2.

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