Figure 4.
Lysosomal SOD1 is active and crucial for lysosomal ROS clearance. (A) Immunoblotting shows that compared with control (Ctrl) 293T cells, the levels of endogenous SOD1 remain unchanged under starvation (Strv) or starvation plus Baf treatment. (B) Recombinant SOD1, purified from E. coli, exhibits enzymatic activity at both pH 7.2 and pH 5.2. The SOD activity of SOD1 at pH 5.2 is ∼20% of its activity at pH 7.2. Quantification of SOD activity is presented as mean ± SEM (n = 3). ***, P < 0.001; ****, P < 0.0001. (C) SOD enzyme activity is detected in purified lysosomes. The activity is significantly increased upon starvation and suppressed by siSOD1 or siTP53INP1. Quantification of SOD activity is presented as mean ± SEM (n = 3). ****, P < 0.0001. (D and E) Compared with NC 293T cells under normal conditions (Ctrl), the lysosomal superoxide levels detected by the HKSOX-2L probe are increased after starvation (Strv) and further elevated by siSOD1 or siTP53INP1 (D). Quantification of HKSOX-2L intensity is presented as mean ± SEM (NC Ctrl, n = 20; NC Strv, n = 20; siSOD1 Strv, n = 21; siTP53INP1 Strv, n = 21) (E). ****, P < 0.0001. Bars: 5 μm. (F and G) Confocal images show that lysosomal superoxide levels detected by the HKSOX-2L probe are increased after starvation (Strv) and further elevated by siTP53INP1. The siTP53INP1-induced elevation is rescued by the overexpression of WT RNAi-resistant SNAP-TP53INP1r, but not SNAP-TP53INP1(ΔLIR)r or SNAP-TP53INP1(ΔC)r (F). Quantification of HKSOX-2L intensity is presented as mean ± SEM (NC + SNAP Ctrl, n = 20; NC + SNAP Strv, n = 20; siTP53INP1 + SNAP-TP53INP1r Strv, n = 20; siTP53INP1 + SNAP-TP53INP1(ΔLIR)r Strv, n = 20; siTP53INP1 + SNAP-TP53INP1(ΔC)r Strv, n = 20) (G). ****, P < 0.0001. Bars: 5 μm. Source data are available for this figure: SourceData F4.

Lysosomal SOD1 is active and crucial for lysosomal ROS clearance. (A) Immunoblotting shows that compared with control (Ctrl) 293T cells, the levels of endogenous SOD1 remain unchanged under starvation (Strv) or starvation plus Baf treatment. (B) Recombinant SOD1, purified from E. coli, exhibits enzymatic activity at both pH 7.2 and pH 5.2. The SOD activity of SOD1 at pH 5.2 is ∼20% of its activity at pH 7.2. Quantification of SOD activity is presented as mean ± SEM (n = 3). ***, P < 0.001; ****, P < 0.0001. (C) SOD enzyme activity is detected in purified lysosomes. The activity is significantly increased upon starvation and suppressed by siSOD1 or siTP53INP1. Quantification of SOD activity is presented as mean ± SEM (n = 3). ****, P < 0.0001. (D and E) Compared with NC 293T cells under normal conditions (Ctrl), the lysosomal superoxide levels detected by the HKSOX-2L probe are increased after starvation (Strv) and further elevated by siSOD1 or siTP53INP1 (D). Quantification of HKSOX-2L intensity is presented as mean ± SEM (NC Ctrl, n = 20; NC Strv, n = 20; siSOD1 Strv, n = 21; siTP53INP1 Strv, n = 21) (E). ****, P < 0.0001. Bars: 5 μm. (F and G) Confocal images show that lysosomal superoxide levels detected by the HKSOX-2L probe are increased after starvation (Strv) and further elevated by siTP53INP1. The siTP53INP1-induced elevation is rescued by the overexpression of WT RNAi-resistant SNAP-TP53INP1r, but not SNAP-TP53INP1(ΔLIR)r or SNAP-TP53INP1(ΔC)r (F). Quantification of HKSOX-2L intensity is presented as mean ± SEM (NC + SNAP Ctrl, n = 20; NC + SNAP Strv, n = 20; siTP53INP1 + SNAP-TP53INP1r Strv, n = 20; siTP53INP1 + SNAP-TP53INP1(ΔLIR)r Strv, n = 20; siTP53INP1 + SNAP-TP53INP1(ΔC)r Strv, n = 20) (G). ****, P < 0.0001. Bars: 5 μm. Source data are available for this figure: SourceData F4.

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