Journal: The Journal of Biological Chemistry

Article Title: A liquid-to-solid phase transition of Cu/Zn superoxide dismutase 1 initiated by oxidation and disease mutation

doi: 10.1016/j.jbc.2022.102857

Figure Lengend Snippet: SOD1 forms misfolding by oxidative stress in cells and in vitro . A , thio T fluorescence assay of 40 μM apo-SOD1 with or without 200 μM H 2 O 2 . The intensity was normalized with the highest intensity as 100% and the lowest intensity as 0%. n = 3 biologically independent samples, data are presented as mean values ± S.D. B , TEM image of 40 μM, oxidized apo-SOD1. Scale bars represent 500 nm. C , confocal microscopy images of EGFP-SOD1 aggregates ( arrows ) in both N2a and HEK-293T cells after H 2 O 2 treatment (100 μM, 3 h). Scale bar represents 25 μm, 10 μm. D , FRAP of the condensates formed by oxidized SOD1 in HEK-293T cells. The intensity was normalized with the pre-bleached as 100% and the first time point after bleaching as 0%. n = 3 biologically independent samples, data are presented as mean values ± S.D. Scale bar represents 10 μm. FRAP, fluorescence recovery after photobleaching; SOD, superoxide dismutase; TEM, transmission electron microscope.

Article Snippet: FRAP assay was conducted using the FRAP module of the Zeiss LSM 780 confocal microscope using 100 × magnification oil objective.

Techniques: In Vitro, Fluorescence, Confocal Microscopy, Transmission Assay, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: A liquid-to-solid phase transition of Cu/Zn superoxide dismutase 1 initiated by oxidation and disease mutation

doi: 10.1016/j.jbc.2022.102857

Figure Lengend Snippet: H 2 O 2 promotes SOD1 phase separation. A , confocal microscopy images of 100 μM EGFP-SOD1 with 10% PEG. Scale bar represents 10 μm. B , droplet fusion experiment of 100 μM EGFP-SOD1 with 10% PEG. Scale bar represents 10 μm. C , FRAP of the droplets formed by 40 μM EGFP-SOD1 with 10% PEG. The intensity was normalized with the pre-bleached as 100% and the first time point after bleaching as 0%. n = 3 biologically independent samples, data are presented as mean values ± S.D. Scale bar represents 2.5 μm. D , turbidity measurement of 0 to 100 μM EGFP-SOD1 with 0 to 15% PEG. n = 3 biologically independent samples. Data are presented as mean values ± S.D. Data were analyzed by two-way ANOVA with multiple comparisons. E , turbidity measurement of 40 μM EGFP-SOD1 with 0 to 1000 mM NaCl concentrations in the presence of 10% PEG. n = 3 biologically independent samples. Data are presented as mean values ± S.D. Data were analyzed by ordinary one-way ANOVA with multiple comparisons. ns, not significant. F , confocal microscopy images of 20 μM EGFP-SOD1 at 25 °C or 37 °C in the presence of 15% PEG. Scale bar represents 10 μm. G , confocal microscopy images of 40 μM EGFP-SOD1 treated with H 2 O 2 (0–20 mM, incubated for 2 h at room temperature) in the presence of 136 mM NaCl and 10% PEG. Scale bar represents 10 μm. H , difference in turbidity before and after incubation with H 2 O 2 (ranging from 0–20 mM). Δturbidity = (A600 nm at 2 h − A600 nm at 0 h). n = 3 biologically independent samples. Data are presented as mean values ± S.D. Data were analyzed by ordinary one-way ANOVA with multiple comparisons. ns, not significant. I , confocal microscopy images of SOD1(100 μM, 15% PEG) colocalized with 20 μM ThT in the presence or absence of 1 mM H 2 O 2 (at room temperature for 24 h). Scale bar represents 10 μm. J , cytotoxicity of 40 μM SOD1 with or without 200 μM H 2 O 2 measured by CCK-8 method. Cells were incubated with oxidized or unoxidized SOD1 at 37 °C for 3 days, by which time fibrils had formed in the cells. n = 4 biologically independent samples. Data are presented as mean values ± S.D. Data were analyzed by two-way ANOVA with multiple comparisons. ns, not significant. FRAP, fluorescence recovery after photobleaching; SOD, superoxide dismutase; ThT, thioflavin T.

Article Snippet: FRAP assay was conducted using the FRAP module of the Zeiss LSM 780 confocal microscope using 100 × magnification oil objective.

Techniques: Confocal Microscopy, Incubation, CCK-8 Assay, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: A liquid-to-solid phase transition of Cu/Zn superoxide dismutase 1 initiated by oxidation and disease mutation

doi: 10.1016/j.jbc.2022.102857

Figure Lengend Snippet: ALS mutations induce SOD1 protein aggregation. A , confocal microscopy images of condensates formed by 50 μM EGFP-SOD1 or mutants with 10% PEG. Scale bar represents 10 μm. B , circularity quantification of the droplets/condensates in ( A ) via intensity thresholding and region of interest (ROI) auto-selection, circularity = 4π × [Area]/([Perimeter]∧2) ranges from 0 (infinitely elongated polygon) to 1 (positive circle). Dot plot of circularity of all droplets/condensates. n = 3 biologically independent samples, data were analyzed by ordinary one-way ANOVA with multiple comparisons. C , FRAP of the condensates formed by EGFP-G93A. The intensity was normalized with the pre-bleached as 100% and the first time point after bleaching as 0%. n = 3 biologically independent samples, data are presented as mean values ± S.D. Scale bar represents 2.5 μm. D , FRAP of the condensates formed by EGFP-A4V. The intensity was normalized with the pre-bleached as 100% and the first time point after bleaching as 0%. n = 3 biologically independent samples, data are presented as mean values ± S.D. Scale bar represents 2.5 μm. E , quantification of mobile fraction of EGFP-SOD1 or mutants. n = 3 biologically independent samples. Data are presented as mean values ± S.D. Data were analyzed by ordinary one-way ANOVA with multiple comparisons. F , ThT fluorescence assay of 40 μM apo-SOD1 and mutants. n = 3 biologically independent samples. Data are presented as mean values ± S.D. G , TEM images of 40 μM apo-SOD1 and mutants. Scale bars represent 500 nm. H , cytotoxicity of 40 μM SOD1 and mutant aggregates measured by CCK-8 method. Cells were incubated with protein at 37 °C for 3 days. n = 3 biologically independent samples. Data are presented as mean values ± S.D. Data were analyzed by two-way ANOVA with multiple comparisons. ns, not significant. ALS, amyotrophic lateral sclerosis; FRAP, fluorescence recovery after photobleaching; SOD, superoxide dismutase; TEM, transmission electron microscope; ThT, thioflavin T.

Article Snippet: FRAP assay was conducted using the FRAP module of the Zeiss LSM 780 confocal microscope using 100 × magnification oil objective.

Techniques: Confocal Microscopy, Selection, Fluorescence, Mutagenesis, CCK-8 Assay, Incubation, Transmission Assay, Microscopy