Fig. 6

USP8 dynamically regulates the EPG5-LC3 interaction. a Western blotting for LC3 and β-actin in Usp8^+/+ and Usp8^▵/− ESCs. β-Actin served as the loading control. Cells were treated with 50 μM chloroquine (CQ) for 2 h. b Quantification of autophagic flux in a by calculating the ratio of LC3-II with or without CQ treatment. Error bars indicate the SD (n = 3). Three independent biological replicates. **P < 0.01 by unpaired two-tailed Student’s t test. c Western blotting for LC3 and β-actin in WT and Usp8-overexpression stable ESCs upon nutrient deprivation. β-Actin served as the loading control. Cells were treated with 50 μM chloroquine (C) for 2 h and/or starved (S) in EBSS for 2 h. d Quantification of autophagic flux by calculating the ratio of LC3-II with or without CQ treatment in c. Error bars indicate the SD (n = 3). Three independent biological replicates. **P < 0.01 by unpaired two-tailed Student’s t test. e Sequence alignment of the LC3-binding region (LIR) in human, mouse, and C. elegans EPG5 proteins. The aromatic residues in the two LIR motifs are indicated by asterisks. Conserved hydrophilic residues are in gray, conserved hydrophobic residues are in green, and conserved negatively charged residues are in blue. f Lysates of Usp8-overexpression and siUsp8-knockdown ESCs were immunoprecipitated with anti-LC3 or anti-IgG and immunoblotted with anti-EPG5 and anti-IgG antibodies. Input cell lysates were immunoblotted with anti-LC3 and anti-actin antibodies as controls. Cells were starved for 2 h before analysis. g A proposed model of the role of the USP8-EPG5-LC3 pathway in regulating ESC stemness