Fig. 1: De novo-synthesized PC and autophagic membranes.
From: Long-term autophagy is sustained by activation of CCTβ3 on lipid droplets
a Labeling of de novo-synthesized PC. MEFs were cultured for 1 h with 0.25 mM propargylcholine in regular medium (a) or in starvation medium (b), fixed, and reacted with Cy3-azide for fluorescence microscopy. Bar, 10 μm. b De novo-synthesized PC and GFP-tagged Atg proteins in MEFs starved for 1 h. PC (magenta) and Atg proteins (green): GFP-ULK1, GFP-DFCP1, GFP-WIPI1, and GFP-LC3. Endogenous EEA1 is labeled as a control. Bar, 10 μm. The box plot shows the ratio of Atg protein (and EEA1) pixels colocalizing with PC puncta. Pooled data from three independent experiments (n > 130); *p = 0.0042, ****p < 0.0001 (two-tailed Mann–Whitney test). c Double labeling of de novo-synthesized PC (magenta) and endogenous LC3 (green) in MEFs starved for 1 h. Bar, 10 μm. d Labeling of existing PC. MEFs were cultured for 1 h with 0.25 mM propargylcholine in regular medium and then for the next 1 h with 5 mM choline in starvation medium. Bar, 10 μm. The total area and the number of PC puncta in a cell were measured. Pooled data from three independent experiments (n = 40); *p = 0.0367, ***p = 0.0002 (two-tailed Mann–Whitney test). In box plots, the center line indicates the median, box boundaries indicate the 25th and 75th percentiles, whiskers are Tukey-type, and the average is marked as +. Source data are provided as a Source data file.
