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. 2022 Oct 27;41(23):e110771. doi: 10.15252/embj.2022110771

Figure 3. Addition of choline rescues autophagy in Δopi3 cells.

Figure 3

  • A, B
    WT and Δopi3 cells expressing GFP‐Atg8 were grown to log phase in SD‐URA, and shifted to SD‐N for 4 h, choline (1 mM) was not supplemented (−choline) or supplemented to SD and SD‐N (+choline SD, SD‐N) or only to SD‐N (+choline, SD‐N) as indicated. Cells were harvested at indicated time points and subjected to western blotting (left panel). Pgk1 was monitored as a loading control. Middle panel‐ Autophagic activity was quantified during starvation by calculating the ratio of free GFP to total GFP (GFP‐Atg8 + free GFP). Statistical analysis was done by ANOVA multiple comparisons test‐Sidak's, compared with WT (ns, not significant, **P ≤ 0.005, ****P ≤ 0.0001), error bars represent SEM of at least 3 independent experiments. Right panel‐ Ape1 maturation was quantified by measuring the mApe1 level out of the total Ape1 amount in Δopi3 cells at growth (SD) or during starvation (SD‐N), with choline supplemented as indicated. Statistical analysis was done by Sidak's multiple comparisons test compared with WT (ns, not significant, *P ≤ 0.05, **P ≤ 0.005), error bars represent SEM of at least 3 independent experiments.
  • C
    Representative images of GFP‐Atg8 (green) and vacuole (red, stained with FM4‐64). WT and Δopi3 cells expressing GFP‐Atg8 were grown in SD‐URA and shifted to SD‐N. Choline (1 mM) was either excluded (−choline), added to growth and starvation medium (+choline SD, SD‐N) or added only to starvation medium (+choline, SD‐N). Cells were observed using confocal microscopy during starvation (left panel). Scale bar 1 μm. Right panel‐ Quantification of cells with GFP inside vacuoles. Statistical analysis was done by ANOVA multiple comparisons test‐Sidak's, compared with WT (****P ≤ 0.0001, ns, not significant), error bars represent SEM of 3 independent experiments. Number of cells analyzed for each strain condition; Choline: WT (n = 487), Δopi3 (n = 316), Choline SD, SD‐N: WT (n = 251), Δopi3 (n = 361), Choline SD‐N: WT (n = 320), Δopi3 (n = 267).
  • D
    PC levels determined by shotgun lipidomics for WT and Δopi3 cells expressing GFP‐Atg8, with or without choline (1 mM) supplementation to SD‐N. Samples were taken after 4 h in SD‐N medium. Statistical analysis was done by ANOVA multiple comparisons test‐Sidak's (****P ≤ 0.0001, ns‐ not significant), error bars represent SEM of at least 3 independent experiments.

Source data are available online for this figure.