Figure 5: ANT–VDAC1 complex is involved in the nuclear translocation of SSBP1.

(a) Heat shock-mediated PTP opening is inhibited by cyclosporin A. HeLa cells, pretreated for 2 h with 20 μM cyclosporin A/2% ethanol or 2% ethanol, were treated without (Cont.) or with heat shock at 42 °C for 60 min (HS), and were incubated with calcein AM in the presence or absence of CoCl₂. Ionomycin was co-incubated as a negative control. Scale bars, 10 μm. The intensity of calcein fluorescence (arbitrary units) from three independent experiments was quantified using ImageJ. Mean±s.d. is shown. Asterisks indicate P<0.05 by Student’s t-test. (b) Cyclosporin A inhibits the nuclear translocation of SSBP1. HeLa cells treated as described in a were co-stained with an antibodies for SSBP1 (green) and TOM20 (red), and DAPI (blue), and fluorescence images were merged (Merge). Scale bars, 10 μm. (c) Cytoplasmic (C), nuclear (N) and mitochondrial (M) fractions were prepared from cells treated as described in b. Equivalent volumes of the fractions were subjected to western blotting using an antibody for SSBP1, HSP90, SP1 and TOM20. (d) VDAC1 knockdown inhibits the nuclear translocation of SSBP1. HeLa cells were infected with Ad-sh-SCR or adenovirus expressing short hairpin RNA (shRNA) for each VDAC for 72 h, and then treated without (Cont.) or with heat shock at 42 °C for 60 min (HS). These cells were co-stained as described in b. Scale bars, 10 μm. (e) Cells treated as described in d were subjected to subcellular fractionation as described in c. (f) Expression of HSP70 in VDAC knockdown cells. MEF cells were infected with Ad-sh-SCR or adenovirus expressing shRNA for mouse VDAC1, VDAC2 or SSBP1 for 72 h, and heat shocked at 42 °C for the indicated periods. HSP70 mRNA was quantified by RT-qPCR (n=3). Mean±s.d. is shown. Asterisks indicate P<0.05 by analysis of variance. (g) Restoration of HSP70 expression by the overexpression of human SSBP1ΔMTS in VDAC1 knockdown cells. VDAC1 or SSBP1 was knocked down in cells, and then c-myc-tagged hSSBP1ΔMTS was overexpressed. HSP70 mRNA levels during heat shock at 42 °C for 30 min were quantified by RT-qPCR (n=3; left). Mean±s.d. is shown. Asterisks indicate P<0.05 by Student’s t-test. Cell extracts were subjected to western blotting (right).