Fig. 7: CCTβ3 in U2OS.
From: Long-term autophagy is sustained by activation of CCTβ3 on lipid droplets
a Distribution of CCTβ3-mCherry (magenta) and LDs (green). Bar, 10 μm. b The autophagic flux after starvation for 24 h. Wild-type U2OS and CCTβ-null U2OS (left panel), and CCTβ-null U2OS transfected with an empty vector, CCTβ2 cDNA, or CCTβ3 cDNA (right panel) were compared. A representative result of three independent experiments. c The autophagic flux after 2 days of hypoxia (0.1% O2) in regular medium. A representative result of two independent experiments. d Cell survival in prolonged starvation. Cells were plated on day 0 and switched to starvation medium on day 1. The number of living cells on day 1 was set as 100%. Pooled data obtained in two independent experiments. Mean ± s.d. of 12 samples. ****p < 0.0001 (unpaired two-tailed t-test). e Endogenous LC3 in wild-type and CCTβ-null U2OS cultured for 24 h in starvation medium. Bar, 10 μm. Pooled data obtained in three independent experiments (n = 90); ****p < 0.0001 (two-tailed Mann–Whitney test). f Quantification of autophagosomes by EM. Wild-type and CCTβ-null U2OS cultured for 24 h in starvation medium. The average autophagosome size (left) and the total area of autophagosomes in the cytoplasm (right). Pooled data from four independent experiments. ***p = 0.0010 (two-tailed Mann–Whitney test). The number of cells examined: 45 (wild-type) and 41 (CCTβ-null). The number of autophagosomes examined: 249 (wild-type) and 83 (CCTβ-null). g Elongated isolation membrane in CCTβ-null U2OS starved for 24 h. Bar, 0.2 μm. IMAT membranes (arrows). See Supplementary Fig. 7G for additional micrographs. h The numerical ratio of isolation membranes to autophagosomes measured by EM. Wild-type and CCTβ-null U2OS cultured for 24 h in starvation medium. Four independent experiments. *p = 0.0286 (two-tailed Mann–Whitney test). The number of isolation membranes observed: 24 (wild-type) and 21 (CCTβ-null). i CCTβ3 in autophagy. In cells starved for a short time, CCTβ3 is activated near autophagic membranes. After prolonged starvation, CCTβ3 is recruited to LDs generated from autophagic digests, and the resultant activation of PC synthesis facilitates autophagic membrane formation near LDs, thereby sustaining autophagy. 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.
