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. 2021 Oct 29;220(12):e202103030. doi: 10.1083/jcb.202103030

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© 2021 Chandra et al.

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Figure 6. — Atg39-containing NE blebs are delivered to vacuoles by autophagy. (A) Deconvolved fluorescence micrographs of a time course (30-min intervals) of rapamycin- or carrier-alone (DMSO)–treated cells expressing Atg39-GFP (green) in a pep4Δ strain; vacuoles are stained with FM 4-64 (magenta). Merged fluorescent images are shown. Image scale in green channel chosen to saturate the bleb fluorescence and allow visualization of NE. Scale bars are 3 μm. (B) Line plot of percentage of cells where Atg39-GFP is visible in vacuoles after treatment with rapamycin (circles and magenta line) or carrier (squares and blue line) over the time points indicated. 75 cells were evaluated each from three independent replicates. SD from the mean percentage is indicated by the shaded area. (C) Violin plot of the fluorescence intensity of Atg39-GFP along the nuclear periphery normalized to background fluorescence in the presence of rapamycin (+) or carrier (−) at the indicated time points. 30 cells each from three independent replicates were evaluated; each replicate was normalized to the mean NE fluorescence at 0 min. Solid line denotes the median; width of the violin plot denotes relative frequency of data points. **, P ≤ 0.01; ****, P ≤ 0.0001 by Brown–Forsythe and Welch ANOVA tests with Games–Howell test for multiple comparisons. (D) Western blot (WB) of proteins from whole-cell extracts derived from rapamycin- or carrier-alone (DMSO)–treated cells expressing Atg39-GFP in the indicated strains. GFP detected with anti-GFP.2 antibody, HRP-conjugated secondary antibodies, and ECL. To assess relative protein loading, a portion of the blots are shown stained with Ponceau S. Position of mol wt standards (in kD) at right. (E) Deconvolved fluorescence micrographs of rapamycin- or carrier-alone (DMSO)–treated cells overexpressing mCherry-Atg39 (magenta) and heh1-ΔL-GFP (green) in a pep4Δ strain; vacuoles are labeled with FM 4-64 (magenta). Merged fluorescent images shown. Asterisks denote the nucleus; arrowheads point to colocalization of GFP and mCherry in vacuole. Scale bars are 5 μm. Source data are available for this figure: SourceData F6.