Fig. 2: De novo-synthesized PC observed by freeze-fracture EM.
From: Long-term autophagy is sustained by activation of CCTβ3 on lipid droplets
a De novo-synthesized PC labeling. MEFs cultured for 1 h with 0.25 mM propargylcholine in starvation medium were quick-frozen and processed for EM. (a) The convex and concave fracture faces of autophagosome. Cytoplasmic leaflet (green), non-cytoplasmic leaflet (pink). See Supplementary Fig. 2B for schematic diagram of autophagosomes in freeze-fracture EM. (b) The cytoplasmic leaflet of the ONM. (c) A negative control. Copper sulfate was omitted from the click reaction solution. Bar, 0.2 μm. b PC labeling density. The labels of cytoplasmic and non-cytoplasmic leaflets are combined. The center line indicates the median, box boundaries indicate the 25th and 75th percentiles, whiskers are Tukey-type, and the average is marked as +. Pooled data obtained in three independent experiments. *1p = 0.0203, *2p = 0.0103 (unpaired two-tailed t-test). The total number of areas measured: 49 (autophagosome) and 14 (ONM). The total area measured: 13.11 μm2 (autophagosome) and 23.71 μm2 (ONM). The labeling density in respective membrane leaflets is shown in Supplementary Fig. 2C. c Comparison of PC labeling density in the same cell. Gray lines connect the labeling density in the autophagosome and the ONM in a cell. The medians and the 25th and 75th percentiles are shown by colored circles and error bars. Pooled data obtained in three independent experiments (n = 23). ***p = 0.0003 (two-tailed Wilcoxon signed-rank test). See Supplementary Fig. 2E for a representative set of micrographs that are used for this analysis. Source data are provided as a Source data file.
