Fig. 2: Cellular inclusions of the ectopically expressed SOD1 proteins in SK-N-SH cells.

a The dot blot analysis of the misfolded SOD1 ectopically expressed in SK-N-SH cells. To detect the misfolded SOD1 in the cell lysate, the anti-misfolded SOD1 antibody was used (left). SK-N-SH cells were transfected with pcDNA3.1 plasmid (EV), pcDNA3.1 expressing wild-type SOD1 (SOD1-WT), or pcDNA3.1 expressing C57D/C146D mutant SOD1 (SOD1-C57D/C146D). The Zn²⁺ chelator (TPEN) or the Cu²⁺ chelator (ATN-224) was treated in the cells during SOD1 transfection. Actin was detected by the actin antibody as a loading control (right). b Top, immunofluorescence staining of overexpressed SOD1 in SK-N-SH cells. SK-N-SH cells transfected with pcDNA3.1 expressing the wild-type SOD1 or the C57D/C146D SOD1 gene were incubated with Cu²⁺ chelator (ATN-224, right panels) or Zn²⁺ chelator (TPEN, middle panels), or without any treatment (left panels) for 12 h. Cells were visualized by the appropriate antibody and dyes: SOD1 in green, ER in red, and DNA in blue. Colocalization of SOD1 with ER shows an orange field. Arrows indicate the cytoplasmic accumulation of SOD1 proteins. White arrows indicated cells with SOD1 inclusions. Scale bar; 10 μm. Bottom, statistical quantification of the inclusion positive cells detected by immunofluorescence staining. The percentages of inclusion positive cells to the total cells were represented in bar graphs with mean ± SD from seven biological replicates. Statistical comparisons were performed using Student’s t-test. Statistical comparisons were performed using Student’s t-test. P-value < 0.05 was considered significant. *, **, *** denote p < 0.05, p < 0.01 and p < 0.001, respectively.