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.

Fig 3: Nrf2 activation-dependent regulation in raising antioxidation and decreasing mutant ataxin-3 expression. MJD78 cells were transiently transfected with si-NTC or si-Nrf2 and with or without an ARE-luciferase reporter construct for 16 h. They were then treated with or without DMSO vehicle, 1 µM JM17 and 0.3 µM RTA-408 for 24 h, respectively. (A) Protein expression of Nrf2, NQO1, HO-1, SOD1, SOD2 and mutant and normal ataxin3. Levels of (B) ARE luciferase activity, (C) total GSH, (D) reduced GSH and (E) catalase are analyzed after treatments. Data are presented as the mean ±SD of at least three independent experiments. Values from the treated cells were normalized to those of the MJD78 cells treated with si-NTC transfection vehicle only. * p < 0.05, compare to non-treated MJD78 with si-NTC group; # p < 0.05, compare to non-treated MJD78 with si-Nrf2 group.

Fig 4: Western blot analysis of SOD1 expression in mesenteric arteries from mice fed with SD, SD plus treatment with luteolin (10 mg/Kg/day), HFD or HFD plus treatment with luteolin (10 mg/Kg/day) for 8 weeks. The figure displays representative blots and column graph referring to the densitometric analysis of immunoreactive bands of SOD1 normalized to the expression of ß-actin. Each column represents the mean ± SEM of five animals. aP < 0.05, significant difference vs. SD; *P < 0.05 significant difference vs. HFD. Statistics: two-way analysis of variance followed by Newman–Keuls test.

Fig 5: PM aggravated ROS production in endothelial cells treated with high glucose, and 1,25(OH)2D3 pretreatment attenuated this change. HUVECs were pretreated with high glucose (30 mM) for 24 h and then treated with PM (50 µg/mL) for 8 h. Before exposure to PM, HUVECs were pretreated with 100 nM 1,25(OH)2D3 for 12 h. A Mitochondrial and cellular ROS levels were detected by staining HUVECs with MitoSOX Red, DHE and DCFDA and examining them under a fluorescence microscope. Bar = 50 µm. B Mitochondrial and cellular ROS levels were detected by staining HUVECs with MitoSOX Red, DHE and DCFDA and analyzed using flow cytometry. C HUVECs were pretreated with 5 µM 25(OH)D3, 50 or 100 nM 1,25(OH)2D3, 500 nM MitoTEMPO and 10 mM NAC (an ROS scavenger) for 12 h and 1 h, respectively. Mitochondrial ROS was detected using MitoSOX Red and a flow cytometer. D HUVECs were pretreated with 5 µM 25(OH)D3, 50 or 100 nM 1,25(OH)2D3, 500 nM MitoTEMPO and 10 mM NAC for 12 h and 1 h, respectively. The MTT assay was used to evaluate cell viability. E The levels of SOD1 and SOD2 expression were detected using Western blotting. *P < 0.05 compared with the control group; †P < 0.05 compared with the HG group; #P < 0.05 compared with the PM group; §P < 0.05 compared with the HG + PM group