Fig. 4

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