Figure S3. SOD1 is important for... | Rockefeller University Press

Figure S3.

$SOD1 is important for maintaining lysosomal homeostasis. (A and B) Confocal images of 293T cells expressing THIO-GFP-mCherry under control (Ctrl), starvation (Strv), starvation plus wortmannin (Wort), or starvation plus Baf conditions (A). Quantification of mCherry-only puncta is presented as mean ± SEM (Ctrl, n = 26; Strv, n = 25; Strv + Wort, n = 30; Strv + Baf, n = 31) (B). ****, P < 0.0001. Bars: 5 μm; insets, 2 μm. (C and D) Confocal images of 293T cells expressing THIO-GFP-mCherry and LAMP1-BFP under starvation (Strv) conditions (C). (D) shows relative fluorescence intensity plots along the red dotted line in the inset in C. Bars: 5 μm; inset, 2 μm. (E) THIO-Flag is detected in purified lysosomal fractions. Levels of THIO-Flag in lysosomes are increased after starvation (Strv) compared with control conditions (Ctrl). (F) Catalase enzyme activity is detected in purified lysosomes from cells under control (Ctrl) and starvation (Strv) conditions. Quantification of catalase activity is presented as mean ± SEM (n = 3). ***, P < 0.001. (G) Recombinant THIO, purified from E. coli, exhibits enzymatic activity at both pH 7.2 and pH 5.2. The catalase activity of THIO at pH 5.2 is similar to its activity at pH 7.2. Quantification of catalase activity is presented as mean ± SEM (n = 3). (H) In a GFP-Trap assay, mCherry-TP53INP1 is immunoprecipitated by THIO-GFP. (I) Confocal images of NC and siTP53INP1 293T cells expressing THIO-GFP-mCherry under control (Ctrl) or starvation (Strv) conditions. Bars: 5 μm; insets, 2 μm. (J and K) DQ-BSA assays show that the DQ-BSA intensity upon starvation is significantly decreased in siSOD1 or siTP53INP1 cells (J). Quantification of DQ-BSA intensity is presented as mean ± SEM (NC, n = 21; siSOD1, n = 19; siTP53INP1, n = 22) (K). Bars: 5 μm. Dotted circles indicate individual cells. **, P < 0.01; ****, P < 0.0001. Bar: 5 μm. (L–N) Confocal images of 293T cells treated with NC, siSOD1, or siTP53INP1 and stained with anti-LAMP1 under starvation (Strv) conditions (L). Cumulative frequency distributions of lysosomal diameters in NC, siSOD1 and siTP53INP1 cells (NC, n = 307 LAMP1-positive puncta from 20 cells; siSOD1, n = 295 LAMP1-positive puncta from 20 cells; siTP53INP1, n = 300 LAMP1-positive puncta from 19 cells) are shown in M. Percentages of lysosomes with the indicated diameters are shown in N. Compared with NC cells, ***, P < 0.001; ****, P < 0.0001. Bars: 5 μm; insets, 2 μm. (O) Flow cytometry analysis shows that cytosolic ROS levels detected by DCFH probe are increased in siTP53INP1 cells, and this increase is rescued by the overexpression of WT SNAP-TP53INP1r or SNAP-TP53INP1(ΔC)r. (P) Immunoblotting results show that the lysosomal protein CTSL is present in the vesicle fraction used for NAG assays in Fig. 5 D. CTSL, cathepsin L. (Q and R) Confocal images of fed (Ctrl) and starved (Strv) 293T cells transfected with IST1-GFP and treated with 400, 600, and 800 μM LLoMe for 30 min (Q). Quantification of IST1-GFP puncta is presented as mean ± SEM (400 μM Ctrl, n = 20; 400 μM Strv, n = 20; 600 μM Ctrl, n = 20; 600 μM Strv, n = 20; 800 μM Ctrl, n = 20; 800 μM Strv, n = 19) (R). ****, P < 0.0001. Bars: 5 μm. (S and T) Confocal images of starved (Strv) 293T cells transfected with IST1-GFP and treated with 400 μM LLoMe for 30 min. Compared with NC cells, IST1-GFP–positive puncta are present in siSOD1-treated cells after starvation (Strv) (S). Quantification of IST1-GFP puncta is presented as mean ± SEM (NC, n = 19; siSOD1, n = 20; siTP53INP1, n = 20) (T). ***, P < 0.001; ****, P < 0.0001. Bars: 5 μm. LLoMe, L-leucyl–L-leucine methyl ester. Source data are available for this figure: SourceData FS3.$

SOD1 is important for maintaining lysosomal homeostasis. (A and B) Confocal images of 293T cells expressing THIO-GFP-mCherry under control (Ctrl), starvation (Strv), starvation plus wortmannin (Wort), or starvation plus Baf conditions (A). Quantification of mCherry-only puncta is presented as mean ± SEM (Ctrl, n = 26; Strv, n = 25; Strv + Wort, n = 30; Strv + Baf, n = 31) (B). ****, P < 0.0001. Bars: 5 μm; insets, 2 μm. (C and D) Confocal images of 293T cells expressing THIO-GFP-mCherry and LAMP1-BFP under starvation (Strv) conditions (C). (D) shows relative fluorescence intensity plots along the red dotted line in the inset in C. Bars: 5 μm; inset, 2 μm. (E) THIO-Flag is detected in purified lysosomal fractions. Levels of THIO-Flag in lysosomes are increased after starvation (Strv) compared with control conditions (Ctrl). (F) Catalase enzyme activity is detected in purified lysosomes from cells under control (Ctrl) and starvation (Strv) conditions. Quantification of catalase activity is presented as mean ± SEM (n = 3). ***, P < 0.001. (G) Recombinant THIO, purified from E. coli, exhibits enzymatic activity at both pH 7.2 and pH 5.2. The catalase activity of THIO at pH 5.2 is similar to its activity at pH 7.2. Quantification of catalase activity is presented as mean ± SEM (n = 3). (H) In a GFP-Trap assay, mCherry-TP53INP1 is immunoprecipitated by THIO-GFP. (I) Confocal images of NC and siTP53INP1 293T cells expressing THIO-GFP-mCherry under control (Ctrl) or starvation (Strv) conditions. Bars: 5 μm; insets, 2 μm. (J and K) DQ-BSA assays show that the DQ-BSA intensity upon starvation is significantly decreased in siSOD1 or siTP53INP1 cells (J). Quantification of DQ-BSA intensity is presented as mean ± SEM (NC, n = 21; siSOD1, n = 19; siTP53INP1, n = 22) (K). Bars: 5 μm. Dotted circles indicate individual cells. **, P < 0.01; ****, P < 0.0001. Bar: 5 μm. (L–N) Confocal images of 293T cells treated with NC, siSOD1, or siTP53INP1 and stained with anti-LAMP1 under starvation (Strv) conditions (L). Cumulative frequency distributions of lysosomal diameters in NC, siSOD1 and siTP53INP1 cells (NC, n = 307 LAMP1-positive puncta from 20 cells; siSOD1, n = 295 LAMP1-positive puncta from 20 cells; siTP53INP1, n = 300 LAMP1-positive puncta from 19 cells) are shown in M. Percentages of lysosomes with the indicated diameters are shown in N. Compared with NC cells, ***, P < 0.001; ****, P < 0.0001. Bars: 5 μm; insets, 2 μm. (O) Flow cytometry analysis shows that cytosolic ROS levels detected by DCFH probe are increased in siTP53INP1 cells, and this increase is rescued by the overexpression of WT SNAP-TP53INP1^r or SNAP-TP53INP1(ΔC)^r. (P) Immunoblotting results show that the lysosomal protein CTSL is present in the vesicle fraction used for NAG assays in Fig. 5 D. CTSL, cathepsin L. (Q and R) Confocal images of fed (Ctrl) and starved (Strv) 293T cells transfected with IST1-GFP and treated with 400, 600, and 800 μM LLoMe for 30 min (Q). Quantification of IST1-GFP puncta is presented as mean ± SEM (400 μM Ctrl, n = 20; 400 μM Strv, n = 20; 600 μM Ctrl, n = 20; 600 μM Strv, n = 20; 800 μM Ctrl, n = 20; 800 μM Strv, n = 19) (R). ****, P < 0.0001. Bars: 5 μm. (S and T) Confocal images of starved (Strv) 293T cells transfected with IST1-GFP and treated with 400 μM LLoMe for 30 min. Compared with NC cells, IST1-GFP–positive puncta are present in siSOD1-treated cells after starvation (Strv) (S). Quantification of IST1-GFP puncta is presented as mean ± SEM (NC, n = 19; siSOD1, n = 20; siTP53INP1, n = 20) (T). ***, P < 0.001; ****, P < 0.0001. Bars: 5 μm. LLoMe, L-leucyl–L-leucine methyl ester. Source data are available for this figure: SourceData FS3.