Figure 1.

A chemical screen for mitophagy inducers. (A) Diagram of tandem-tagged mCherry-GFP-FIS1_101-152-based mitophagy assay. (B) Co-localization of mCherry-GFP-FIS1_101-152 in U2OS cells with ATP synthase. (C) Screen for mitophagy inducing conditions using the tandem-tag mitophagy assay in SH-SY5Y cells. All treatments for 24 h. For this experiment, quantitation of mitophagy was performed with counter blinded to condition. (D) Example micrographs from cells expressing the mitophagy construct treated under control conditions or with 1 mM DFP for 24 h. (E) Results of mitophagy assay in SH-SY5Y cells following treatment with 1 mM DFP or deferoxamine for 24 h, 340 μM ferric ammonium citrate (iron) was preincubated with 1 mM DFP for 10 min before addition to cells. 100 nM bafilomycin A1 was added for final 4 h of incubation. (F) Representative blot and (G) Quantitation of transferrin receptor protein levels (left axis) and results of tandem-tag mitophagy assay (right axis) from SH-SY5Y cells treated with DFP at indicated concentration or 1 mM DFO for 24 h. All results are from 3–4 independent experiments. All quantitative data are mean±s.e.m. Scale bars, 10 μm. **P<0.01, ***P<0.001, NS, not significant. Baf, bafilomycin A1; DFO, deferoxamine; DFP, deferiprone; GFP, green fluorescent protein.

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