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. 2022 Apr 11;221(5):e202109084. doi: 10.1083/jcb.202109084

Figure S1.

Figure S1.

Relocalization of SE-specific proteins to class E compartments of vps4 mutant cells. (A) Quantification of Ivy1, Tor1, Ego1, and Vps4 dots per cell. Cells (n ≥ 200), Ivy1 dots (n ≥ 200), Tor1 dots (n ≥ 100), Ego1 dots (n ≥ 100), and Vps4 dots (n ≥ 600) were analyzed. Error bars represent SD of three independent experiments. (B) Quantification of endosomal protein puncta relative to Ivy1 (related to Fig. 2 C). Cells (n ≥ 150), Ivy1 dots (n ≥ 150), Vps4 dots (n ≥ 300), Vps8 dots (n ≥ 50), Vps21 dots (n ≥ 200), Ypt7 dots (n ≥ 150), Ego1 dots (n ≥ 50), Gtr2 dots (n ≥ 50), Kog1 dots (n ≥ 50), or Fab1 dots (n ≥ 50) were quantified by ImageJ and are shown as the percentage of endosomal markers that show Ivy1-positive signals. Error bars represent SD of three independent experiments. (C) Quantification of endosomal protein puncta colocalizing with Ivy1 in wild-type (wt) and vps4∆ mutant (related to Fig. 3 D). Cells (n ≥ 200), Ivy1 dots (n ≥ 200), Vps4 dots (n ≥ 400), Vps21 dots (n ≥ 300), Ypt7 dots (n ≥ 100), Ego1 dots (n ≥ 50), Kog1 dots (n ≥ 100), or Fab1 dots (n ≥ 50) were quantified by ImageJ and are expressed as percentage of endosomal markers that show Ivy1-positive signals. Error bars represent SD of three independent experiments. ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01 (Student’s t test). (D) Localization of Ivy1 relative to Fab1 in vps4 ts cells. vps4 ts cells expressing mCherry-tagged Ivy1 and mNeon-tagged Fab1 were grown at 24°C in a synthetic medium, and then shifted or not to 37°C for 1 h. Arrows show colocalizing dots. (E) Quantification of mean fluorescent intensity of Ivy1-mGFP from C. Cells (n ≥ 200) and Ivy1 dots (n ≥ 200) were quantified by ImageJ. Error bars represent SD of three independent experiments. ***, P ≤ 0.001 (Student’s t test). (F) Percentage of Ivy1 colocalizing with Fab1 puncta (upper panel) and percentage of Fab1 puncta colocalizing with Ivy1 (lower panel). Cells (n ≥ 200), Ivy1 dots (n ≥ 200), and Fab1 dots (n ≥ 50) were quantified by ImageJ. Error bars represent SD of three independent experiments. ns, P > 0.05 (Student’s t test). (G) Localization of Ivy1 relative to Cps1. Wild-type and vps4Δ cells expressing mCherry-tagged Ivy1 and GFP-tagged Cps1 were grown in a synthetic medium and analyzed by fluorescence microscopy. Individual slices are shown. Scale bar, 5 µm. Arrows show colocalizing dots. (H) Quantification of Ivy1 colocalizing with Cps1-positive puncta. Cells (n ≥ 150), Ivy1 dots (n ≥ 150), and Cps1 dots (n ≥ 50) were quantified by ImageJ. Error bars represent SD of three independent experiments. **, P ≤ 0.01 (Student’s t test).