Figure 3.

EMC6 promotes autophagic flux in GBM cells. (a and c) The indicated cell lines with or without EMC6 overexpression (or EMC6 knockdown) were infected with Ad5–GFP–LC3 at 50 MOI for 20 h, then treated in the presence or absence of CQ (25 μM) for 2 h. Representative confocal microscopy images of GFP–LC3B distribution obtained from the indicated cell lines are shown (b and d). Quantification of GFP–LC3B puncta per cell treated as in (a) and (c). Data are means±S.D. of at least 100 cells scored. ***P<0.001. (e and g) U87 cells with or without EMC6 overexpression (or EMC6 knockdown) were cultured and treated in the presence or absence of CQ (25 μM) for 2 h. The levels of LC3B-II were detected by western blot. (f and h) Quantification of LC3B-II levels relative to ACTB in cells treated as (e) and (g). Average value in vector group without CQ treatment was normalized as 1. Data are means±S.D. of results from three experiments, *P<0.05, **P<0.01. (i and k) The levels of SQSTM1 in the indicated cell lines with or without EMC6 overexpression (or EMC6 knockdown) were analyzed by western blot. (j and l) Quantification of amounts of SQSTM1 relative to ACTB in cells treated as in (i) and (k). The average value in vector group was normalized as 1. Data are means±S.D. of results from three experiments, **P<0.01. (m and n) The indicated cells were electrotransfected with polyQ80-Firefly luciferase (or control polyQ19-Firefly luciferase) for 24 h. The luminescent signals were detected and polyQ80-luciferase/polyQ19-luciferase ratios were calculated in each group. Data are means±S.D. of results from three experiments, **P<0.01