Fig. 2

Hsp70-Bim PPI mediates stress-induced autophagy rather than basal autophagy. A In-gel silver staining of the proteins enriched by S1g-2-probe, S1-probe and S1n-probe respectively from HEK293T cells cultured in HBSS for 4 h (top); the Hsp70, Bcl-2 and Mcl-1 were further verified by Western blotting (bottom). B Co-IP analysis of Hsp70 interactions with Bim and Beclin 1 in HEK293T cells treated with HBSS in the presence or absence of indicated concentrations of S1g-2 and S1, respectively, and Western blot analysis of LC3 and p62 levels in cell lysates. An equivalent of DMSO were added to the compound untreated group as vehicle control. The top graph shows (mean ± SD, n = 3 biologically independent experiments) the relative level of Bim and Beclin 1 in co-IP of each treatment normalized to that in control cells. The bottom graph shows (mean ± SD, n = 3) LC3-II/LC3-I ratios of each treatment normalized to the LC3-II/LC3-I ratio of control cells. *P < 0.05, **P < 0.01 (one-way ANOVA test). C Western blot analysis of the LC3 level in NS shRNA-transfected, Bim shRNA-transfected and Hsp70 shRNA-transfected HEK293T cells respectively with HBSS treatment in the presence or absence of 10 μM S1g-2 for 4 h. An equivalent of DMSO were added to the compound untreated group as vehicle control. **P < 0.01 (one-way ANOVA test); n.s. indicates no significance. D Western blot analysis of the levels of LC3 and p62 in HEK293T cells treated with 30 nM BafA1, 10 μM S1g-2 and 10 μM S1 respectively for 4 h. ^**P < 0.01 (one-way ANOVA test); n.s. indicates no significance. E Western blot analysis of the levels of LC3 in HEK293T cells treated with HBSS or 100 nM BafA1 in the presence or absence of 10 μM S1g-2 for 4 h. The graphs show (mean ± SD, n = 3 biologically independent experiments) LC3-II/LC3-I ratios normalized to that in control cells. **P < 0.01 (one-way ANOVA test); n.s. indicates no significance