Fig. 2.

Effects of ectopic expression of SQSTM1 mutants on the autophagic activity in mouse embryonic fibroblast. (A) Western blot analysis of SQSTM1 and LC3. MEFs prepared from Sqstm1-KO mice were transfected with pCIneoFLAG-SQSTM1 constructs. After 24 h, the cells were incubated in DMEM or EBSS with or without 250 μM chloroquine (CQ) for additional 6 h. Untransfected MEFs from wild-type (MEF_WT) and Sqstm1-KO (MEF_KO) mice were also treated in the same manner. Two μg of total proteins from each sample was subjected to SDS-PAGE and analyzed by immunoblotting using anti-SQSTM1 (p62-C) and anti-LC3 antibodies. β-actin was used for a loading control. Representative images are shown. Positions of SQSTM1, LC3-I and -LC3-II are shown on the right. Size-markers are shown on the left. (B) Quantification of the signal intensities of SQSTM1 in western blots. Values of SQSTM1/β-actin (in arbitrary unit; AU) are expressed as mean relative to MEF_WT at 0 h [± S.E.M. (n = 3 biological replicates)]. Individual data points are also shown. (C) Quantification of the signal intensities of LC3 in western blots. Values of the ratio of LC3-II/LC3-I + LC3-II are expressed as mean ± S.E.M. (n = 3 biological replicates). Individual data points are also shown. (D) Autophagy flux in MEFs expressing SQSTM1 variant/mutant. The autophagy flux was defined as; [(the ratio with CQ) – (the ratio without CQ)]. Values (in arbitrary unit; AU) are expressed as mean relative to MEF_WT in DMEM [± S.E.M. (n = 3 biological replicates)]. Individual data points are also shown. Statistical significances were evaluated by one-way ANOVA with Tukey's multiple comparison test (*p < 0.05, **p < 0.01, ***p < 0.001) (B and C) or with Dunnett's multiple comparison test (*p < 0.05) (D).