Fig 2: IDH inhibits oxidative stress-induced cell death in DJ-1 null mammalian DA cells.(A) Comparison of IDH1 mRNA expression levels in wild type (WT) and DJ-1 null (DJ-1-/-) SN4741 cells under control (CON) or H2O2 (H2O2) treatment (n = 3). (B) IDH2 mRNA expression level change was analyzed in DJ-1 null SN4741 cells under control (CON) or H2O2 (H2O2) treatment, with scramble (-) or Keap1 siRNA (+) (n = 3). (C) Cell viability of wild type (WT), DJ-1 null (DJ-1-/-), IDH1-overexpressing DJ-1 null (IDH1 DJ-1-/-) and IDH2-overexpressing DJ-1 null (IDH2 DJ-1-/-) SN4741 cells under 1.5 mM H2O2 treatment for 6 h. Cell viability was measured by MTT assay. The inset shows immunoblot of FLAG-tagged IDH1 and IDH2. ß–tubulin as a loading control. (D) Propidium iodide and annexin V FITC staining of H2O2-treated SN4741 cells. (E) Necrotic cell death rates (n = 3). (F) Flow cytometric analysis of CM-H2DCFDA-stained SN4741 cells. Gray-filled area: WT or DJ-1 null SN4741 cells without H2O2 treatment; Red line: H2O2-treated SN4741 cells; Green line: H2O2-treated IDH1-overexpressing SN4741 cells; Blue line: H2O2-treated IDH2-overexpressing SN4741 cells. (G) Fold change in the mean of fluorescence intensity (MFI) of CM-H2DCFDA in flow cytometric analysis (n = 3). (H) Flow cytometric analysis of MitoSOX-stained SN4741 cells. Gray-filled area: WT or DJ-1 null SN4741 cells without H2O2 treatment; Red line: H2O2-treated SN4741 cells; Green line: H2O2-treated IDH1-overexpressing SN4741 cells; Blue line: H2O2-treated IDH2-overexpressing SN4741 cells. (I) Fold change in the mean of fluorescence intensity (MFI) of MitoSOX in flow cytometric analysis (n = 3). (J) Cell viability of MitoTEMPO-treated SN4741 cells under H2O2 treatment. Cell viability was measured by MTT assays (n = 3). Data information: Significance was determined by one-way ANOVA with Sidak correction [**, P<0.01; ***, P<0.001; NS, not significant (P>0.05)]. Error bars indicate SD.

Fig 3: IDH rescues neuronal defects of DJ-1 mutants under oxidative stress.(A) Survival curves of DJ-1ß null mutants (hs DJ-1ßex54), IDHm1-overexpressing DJ-1ß null mutants (hs>IDHm1 DJ-1ßex54), IDHm2-overexpressing DJ-1ß null mutants (hs>IDHm2 DJ-1ßex54), and IDHc-overexpressing DJ-1ß null mutants (hs>IDHc DJ-1ßex54) under rotenone treatment. heat shock (hs)-GAL4/+ (hs) flies were used as controls (log-rank test: P<0.001, n = 120 for each genotype). All life span assays were carried out at 25°C and were repeated at least twice. (B) Comparison of climbing ability of 7-day-old flies after rotenone treatments (n = 5). (C-D) Confocal images (C) and graphs (D) of the average number of DA neurons within DL1, DM, PM, and DL2 clusters of the adult brains from 6-day-old DJ-1ß null mutants (elav DJ-1ßex54), IDHm1-overexpressing DJ-1ß null mutants (elav>IDHm1 DJ-1ßex54), IDHm2-overexpressing DJ-1ß null mutants (elav>IDHm2 DJ-1ßex54) and IDHc-overexpressing DJ-1ß null mutants (elav>IDHc DJ-1ßex54) after rotenone treatments. elav-GAL4/+ (elav) flies were used as controls (n = 40 for elav; n = 37 for elav DJ-1ßex54; n = 36 for elav>IDHm1 DJ-1ßex54; n = 30 for elav>IDHm2 DJ-1ßex54; n = 39 for elav>IDHc DJ-1ßex54). DA neurons were stained with anti-TH antibody (green). Scale bars: 20 µm. (E-G) Comparison of IDHm1 (E), IDHm2 (F), or IDHc (G) mRNA expression levels in the heads and thoraces of wild type (WT) and DJ-1ß null mutants (DJ-1ßex54) under control (CON) or rotenone treatments (Rotenone) (n = 3). Data information: Significance was determined by one-way ANOVA with Sidak correction [*, P<0.05; **, P<0.01; ***, P<0.001; NS, not significant (P>0.05)]. Error bars indicate SD.

Fig 4: TIC protects mammalian DA cells against oxidative stress.(A) NADPH/NADP+ ratio measured in H2O2-treated wild type (WT) and DJ-1 null (DJ-1-/-) SN4741 cells with pre-treatment of TIC at indicated concentrations (n = 3). (B) Cell viability of H2O2-treated SN4741 cells with pre-treatment of TIC at indicated concentrations. Cell viability was measured by MTT assays (n = 3). (C) Propidium iodide and annexin V FITC staining of H2O2-treated SN4741 cells. TIC was pre-treated with indicated concentrations. (D) Necrotic cell death rates (n = 3). (E) Flow cytometric analysis of CM-H2DCFDA-stained SN4741 cells. Gray-filled area: WT or DJ-1 null SN4741 cells without H2O2 treatment; Red line: H2O2-treated SN4741 cells; Green line: TIC and H2O2-treated SN4741 cells. TIC was pre-treated with indicated concentrations. (F) Fold change in the mean of fluorescence intensity (MFI) of CM-H2DCFDA in flow cytometric analysis (n = 3). (G) Flow cytometric analysis of MitoSOX-stained SN4741 cells. Gray-filled area: WT or DJ-1 null SN4741 cells without H2O2 treatment; Red line: H2O2-treated SN4741 cells; Green line: TIC and H2O2-treated SN4741 cells. TIC was pre-treated with indicated concentrations. (H) Fold change in MFI of MitoSOX in flow cytometric analysis (n = 3). (I) Cell viability of H2O2-treated DJ-1 null SN4741cells transfected with control (CON) siRNA, IDH1-specific siRNA (IDH1i) or IDH2-specific siRNA (IDH2i). To downregulate gene expression of both IDHs, IDH1- and IDH2-specific siRNA were co-transfected (IDH1i IDH2i). TIC was pre-treated with indicated concentrations. Cell viability was measured by MTT assays (n = 3). The inset shows immunoblot of endogenous IDH1 and IDH2. ß-tubulin as a loading control. Data information: Significance was determined by one-way ANOVA with Sidak correction (*, P<0.05; **, P<0.01; ***, P<0.001; NS, not significant). Error bars indicate SD.

Fig 5: Schematic models of the protective effects of IDH on DA neurons against oxidative stress in DJ-1 null background.