Fig. 3

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