Figure 2.

Knockdown of EVA1A impairs cell autophagy. (a) Representative confocal microscopy images of the GFP–LC3B distribution in U2OS cells transfected with siControl or siEVA1A for 48 h and treated with BafA₁ (10 nM) and/or EBSS for the last 4 h. (b) Quantification of GFP–LC3B puncta in cells treated with reagents as indicated in (a). Data are means±S.D. of at least 100 cells scored. (c) Western blot analysis of endogenous LC3B-II levels in U2OS cells treated as in (a). (d) Quantification of amounts of LC3B-II relative to ACTB in cells treated as in (c). The average value in siControl transfected cells with BafA₁ treatment was normalized as 1. Data are means±S.D. of results from three experiments. (e) HeLa cells stably expressing GFP–LC3B were transfected with siControl or siEVA1A for 48 h. Levels of free GFP and SQSTM1 were analyzed by western blot. (f) Quantification of the amount of free GFP or SQSTM1 relative to ACTB in cells treated as in (e). The average value in siControl transfected cells was normalized to 1. Data are means±S.D. of results from three experiments. (g) Western blot analysis of endogenous LC3B-II levels in MEFs treated as indicated. (h) Quantification of amounts of LC3B-II relative to ACTB in cells treated as in (g). The average value in Eva1a^+/+ MEF cells without any treatment was normalized as 1. Data are means ± S.D. of results from three experiments. (i) Representative confocal microscopy images of endogenous LC3B distribution in MEFs treated with BafA₁ (10 nM) and/or EBSS for 4 h. (j) Quantification of endogenous LC3B puncta in mef cells as indicated in (i). Data are means ± S.D. of at least 100 cells scored. *P<0.05, *** P<0.001