Additional file 1 of Parkin coregulates glutathione metabolism in adult mammalian brain

Additional file 1: Fig. S1. Wild-type parkin may contribute to lowering of oxidative stress in murine hearts.a Relative H2O2 concentration and b total protein nitrotyrosination in heart homogenates of 6 mth-old mice fromthe indicated genotypes. Ponceau S was used as a loading control for relative signal quantification, as shown in c(n=3/genotype ± SEM). Significance was tested using 1-way ANOVA with Tukey’s post-hoc analysis (a, c); nosignificance was found Fig. S2.. Parkin over-expression alters the redox state in mammalian cells. a Cytotoxicity assayed in CHO cells stably expressing myc-parkin cDNA (denoted as PRKN+) or myc-control vectors (denoted as PRKN-) under normal conditions with or without the addition of 2 mM H2O2 or 20 mM buthionine sulfoximine (BSO). Data in a are plotted as mean normalized to wells of untreated control cells. b Relative endogenous H2O2 levels and c cellular toxicity in CHO cells stably expressing myc-parkin (PRKN+) or myccontrol vector (PRKN-) with or without exposure to 2 mM H2O2, 2 mM BSO, or 20 mM N-acetyl cysteine (NAC), as indicated. d, f HPLC-based quantification of reduced glutathione (GSH), oxidized glutathione (GSSG), the ratio of GSH:GSSG, and total glutathione pool (GSH+2GSSG) in CHO cells under control conditions (d), and following H2O2 stress (f). e Quantification of GSH by monochlorobimane assay (Tietze method) in SH-SY5Y neural cells transiently over-expressing FLAG-parkin (PRKN+) or FLAG-control vector (PRKN-). Results were obtained using 3 independent experiments ± SEM. Statistical significance was determined using a One-sample T-test with each column compared to 1.0 (a; not significant), 2-way ANOVA with Tukey’s post-hoc (b, c) and unpaired Student T-test (d–f), where *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001, as indicated Fig. S3.. Prkn expression alters glutathione metabolism in murine brain. a–d Quantification of reduced glutathione (GSH), oxidized glutathione (GSSG), their ratio, as well as the total concentration of glutathione in brain homogenates from 6 mth-old mice, as quantified by HPLC, for the indicated genotypes. e Glutathione reductase (GR) activity in homogenates of previously frozen brain from 7 to 8 mth-old WT and prkn−/− mice (n = 6, mean normalized to WT ± SEM). Significance was determined using an unpaired Student T-test with p = 0.029. f GR activity measured in freshly prepared brain homogenates of 6 mth-old mice from 4 different genotypes (as in panels a-d; n = 3/genotype). Significance was determined using a 1-way ANOVA with Tukey’s post-hoc analysis (a–d, f) and unpaired Student T-test (e) with *p ≤ 0.05 and **p ≤ 0.01 Fig. S4. Western blot results for redox state-related proteins in fractionated human cortices. a Specimens from matched control and human ARPD (PRKN mutant) frontal cortices, as described above (and in: Shimura H et al. 2001; Tokarew J et al., 2021), were serially fractioned using increasing concentrations of detergent from readily saline-soluble (TS) state, to lipid-bound, TX100-soluble (TX) state and insoluble (SDS) state, as described in Tokarew J et al., 2021. Fractions were analyzed by SDS/PAGE under reducing conditions and immunoblotted with antibodies to parkin, DJ-1, voltage-dependent anion channel (VDAC), manganese superoxide dismutase (MnSOD) and glyoxalase-1, and membranes counterstained with Ponceau S, as indicated.

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