MAGIC regulators revealed by a genome-wide screen in yeast and validations in human RPE-1 cells.
(A) Workflow of the spGFP-based genetic screen in yeast. (B) KEGG pathway analysis of validated mutants that affect MAGIC. The size of the node indicates the number of genes identified. Pathways with at least two associated genes are shown. (C, D) Representative images (C) and quantification (D) of Lsg1 spGFP signal in WT and Δsnf1 cells at 30 °C. Shown in (C): Top, Lsg1 spGFP; Bottom, merged images of spGFP and mitochondria labeled with MTS-mCherry. Shown in (D): means ± SEM of spGFP/mCherry ratio. Unpaired two-tailed t-test. (E, F) Representative images (E) and quantification (F) of Lsg1 spGFP signal in Δltv1 and wild-type LTV1 cells at 30 °C and after HS. Shown in (F): means ± SEM of spGFP/mCherry ratio. Paired two-tailed t-test. HS: heat shock. (G, H) Representative images (G) and quantification (H) of FlucSM spGFP signals in WT (REG1) cells in HG or LG, and Δreg1 cells in HG. Shown in (G): Top, FlucSM spGFP; Bottom, merged images of spGFP and mitochondria labeled with Tom70-mCherry. Shown in (H): means ± SEM of spGFP intensity. Paired (REG1 in HG vs. LG) or unpaired (REG1 vs. Δreg1 in HG) two-tailed t-test. (I) Schematic diagram of Snf1 activation in yeast. (J) Representative images of FlucDM spGFP in RPE-1 cells treated with DMSO, dorsomorphin, or AICAR. Top, FlucDM spGFP; Middle, mitochondria-targeted mCherry; Bottom, merged images. (K-M) Flow cytometry-based quantifications of FlucDM spGFP in RPE-1 cells treated with DMSO, dorsomorphin, or AICAR (K, M), and GST spGFP in cells treated with DMSO or dorsomorphin (L). Means ± SEM of spGFP intensities are shown. Paired two-tailed t-test. **P < 0.01; ***P < 0.001; ns, not significant, P > 0.05. HG: 2% glucose; LG: 0.1% glucose plus 3% glycerol. Scale bars, 5 μm.