Figure S2.
4 nM AntA does not arrest mitochondria by the PINK1/Parkin pathway. (A) Western blot of cortical neurons treated for 5 h with vehicle or 10 µM AntA and 1 µM oligomycin or 1 h of 4 nM AntA. Blots were probed for phospho-S65 ubiquitin or total ubiquitin. Anti-vinculin is shown as a loading control. (B) Quantification of phospho-S65 ubiquitin levels as in A; n = 4 biological repeats. (C) Quantification of mitochondrial membrane potential in hippocampal neurons stained with the reporter dye TMRM. Neurons were pretreated for 1 h with the indicated concentration of ETC inhibitor, after which TMRM and Hoechst were added. Cells were imaged live with both dyes and inhibitor present. Five cell fields were imaged from four independent biological repeats. TMRM intensity is normalized to cell count. (D) Quantification of TMRM intensity in fibroblasts grown in galactose media. Cells were pretreated for 1 h with the indicated concentration of ETC inhibitor, after which TMRM and Hoechst were added. Cells were imaged live with both dyes and indicated ETC inhibitors. 15–20 cell fields were imaged from 4 independent biological repeats. (E) Representative images from neurons treated with vehicle, 4 nM AntA, or 20 µM CCCP, as in C. (F) Representative images from fibroblasts treated with vehicle, 40 nM AntA, or 20 µM CCCP as in D. (G) Western blot of cortical neurons overexpressing MYC-Miro1 from the hSYN1 promoter. Cells were treated with AntA at the indicated concentrations and for the indicated times. Blots were probed with anti-MYC and with anti-GAPDH as a loading control. (H) Quantification of western blots as in G. n = lysates from three independent biological replicates. The P value for B was calculated by a two-tailed, unpaired t test with Welch’s correction. A one-way Welch ANOVA was performed with Dunnett’s T3 multiple comparison correction for C and D. P values were calculated by performing a blocked one-way ANOVA with Dunnett’s T3 multiple comparison correction for H. Scale bars = 20 μm. Source data are available for this figure: SourceData FS2.

4 nM AntA does not arrest mitochondria by the PINK1/Parkin pathway. (A) Western blot of cortical neurons treated for 5 h with vehicle or 10 µM AntA and 1 µM oligomycin or 1 h of 4 nM AntA. Blots were probed for phospho-S65 ubiquitin or total ubiquitin. Anti-vinculin is shown as a loading control. (B) Quantification of phospho-S65 ubiquitin levels as in A; n = 4 biological repeats. (C) Quantification of mitochondrial membrane potential in hippocampal neurons stained with the reporter dye TMRM. Neurons were pretreated for 1 h with the indicated concentration of ETC inhibitor, after which TMRM and Hoechst were added. Cells were imaged live with both dyes and inhibitor present. Five cell fields were imaged from four independent biological repeats. TMRM intensity is normalized to cell count. (D) Quantification of TMRM intensity in fibroblasts grown in galactose media. Cells were pretreated for 1 h with the indicated concentration of ETC inhibitor, after which TMRM and Hoechst were added. Cells were imaged live with both dyes and indicated ETC inhibitors. 15–20 cell fields were imaged from 4 independent biological repeats. (E) Representative images from neurons treated with vehicle, 4 nM AntA, or 20 µM CCCP, as in C. (F) Representative images from fibroblasts treated with vehicle, 40 nM AntA, or 20 µM CCCP as in D. (G) Western blot of cortical neurons overexpressing MYC-Miro1 from the hSYN1 promoter. Cells were treated with AntA at the indicated concentrations and for the indicated times. Blots were probed with anti-MYC and with anti-GAPDH as a loading control. (H) Quantification of western blots as in G. n = lysates from three independent biological replicates. The P value for B was calculated by a two-tailed, unpaired t test with Welch’s correction. A one-way Welch ANOVA was performed with Dunnett’s T3 multiple comparison correction for C and D. P values were calculated by performing a blocked one-way ANOVA with Dunnett’s T3 multiple comparison correction for H. Scale bars = 20 μm. Source data are available for this figure: SourceData FS2.

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