Fig 2: Hsp70-Bim PPI induced mitophagy independent on Bax and Bak. A Western blot analysis of LC3 level in isolated mitochondria from MEFs and Bax/Bak DKO MEFs treated with HBSS 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). B Bax/Bak DKO MEFs transfected with Bim or the respective vector control was treated with 5 mg/mL and 10 mg/mL dox respectively for 48 h, and then the cell lysates were applied for Western blot analysis of Bim. The harvested cells were applied for mitochondria isolation and the LC3 level was analyzed by Western blotting. **P < 0.01 (one-way ANOVA test). C Co-IP analysis of Hsp70 interactions with Bim, TOMM20 and parkin in Bax/Bak DKO MEFs treated as described in B. **P < 0.01 (one-way ANOVA test). D Co-IP analysis of TOMM20 interactions with parkin and TOMM20 ubiquitination which was visualized by Western blot analysis using anti-ubiquitin in Bax/Bak DKO MEFs treated as described in B. All figures represent the results from n = 3 biologically independent experiments. **P < 0.01 (one-way ANOVA test)

Fig 3: 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

Fig 4: Disruption of Hsp70-Bim PPI by S1g-2 inhibits mitophagy in response to various stresses. A Western blots analysis of LC3 level in isolated mitochondria, ER or Golgi from HEK293T cells treated with HBSS 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. Western blot of subcellular fraction probed with antibodies specific for organelle-specific marker proteins: mitochondria (TOMM20), ER (Calnexin), Golgi (GM130). The graphs show (mean ± SD, n = 3 biologically independent experiments) LC3-II/LC3-I ratios of each treatment normalized to the LC3-II/LC3-I ratio of control cells. **P < 0.01 (one-way ANOVA test). B Western blot analysis of TOMM20, Calnexin and GM130 in HEK293T cells treated with H2O2 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. Right panel: relative levels of TOMM20, Calnexin and GM130 normalized to β-actin. The data are expressed as the mean ± SD (n = 3 biologically independent experiments). **P < 0.01 (one-way ANOVA test). C Representative colocalization images of GFP-LC3 (green) and mitochondria (MitoTracker Red). Quantification of the colocalization coefficient between GFP-LC3 and MitoTracker Red displayed as Pearson coefficients in the colocalized volume (1, perfect correlation; 0, no correlation). The data are expressed as the mean ± SD (n = 3 biologically independent experiments). **P < 0.01 (one-way ANOVA test), n = 5–6 dishes, 20 fields per dish. All figures represent the results from n = 3 biologically independent experiments

Fig 5: S1g-2 inhibits the association between TOMM20 and parkin followed by parkin-mediated TOMM20 ubiquitination. A Co-IP analysis of Hsp70 interactions with Bim, TOMM20 and parkin in HEK293T cells treated with 0.5 mM H2O2, 34 μM VP-16 or HBSS 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). B Co-IP analysis of TOMM20 interactions with parkin in HEK293T cells treated as described in A. **P < 0.01 (one-way ANOVA test). Ubiquitinated TOMM20 was visualized by Western blot analysis using anti-ubiquitin. C Representative colocalization images of GFP-parkin expressed HeLa cells treated with H2O2 in the presence or absence of 10 μM S1g-2 for 4 h. Cells were stained for mitochondria (MitoTracker Red). Quantification of the colocalization coefficient between GFP-parkin and MitoTracker Red displayed as Pearson coefficients in the colocalized volume. The data are expressed as the mean ± SD (n = 3 biologically independent experiments). **P < 0.01 (one-way ANOVA test), n = 5–6 dishes, 20 fields per dish. D In vitro ubiquitination assay of TOMM20 in the presence or absence of Hsp70, Bim and S1g-2 respectively or in combination. All figures represent the results from n = 3 biologically independent experiments. E Western blot analysis of PINK1 in HEK293T cells treated with H2O2 in the presence or absence of 10 μM S1g-2 for 4 h. The graphs show relative levels of PINK1 normalized to β-actin. The data are expressed as the mean ± SD (n = 3 biologically independent experiments). **P < 0.01 (one-way ANOVA test); n.s. indicates no significance