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. 2020 Jan 3;16(10):1838–1850. doi: 10.1080/15548627.2019.1709763

Figure 5.

Figure 5.

Double-site mutation of Cys292 and Cys361 compensates for ATG4B impairment upon oxidation and leads to increasing autophagic flux. (A) ATG4B KO HeLa cells stably expressing Flag-ATG4B or Flag-ATG4BC292,361S were stimulated by H2O2 (1 mM) for 1 h, ATG4B KO HeLa cells expressed with empty vector was used as control, corresponding LC3-II level were monitored. (B) Cell lysates (5 µg) from cells in (A) were employed to test ATG4B activity by cleaving LC3-GST substrate, results were represented by CBB analysis. (C) Recombinant His-ATG4B and His-ATG4BC292,361S (0.015 µg) were respectively exposed to air on ice for 2 h, after which enzyme assay was performed. (D-E) ATG4B KO HeLa cells stably expressing Flag-ATG4B or Flag-ATG4BC292,361S growing exponentially in medium were treated with or without CQ (40 µM) for 2 h, followed by immunoblot analysis with anti-LC3B antibody (D), and immunofluorescence using the same antibody (E). N = 44, 49, 54, 47, 51, 55, the number of LC3 puncta were quantified and at least 3 independent experiments were performed. Data are presented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant.