Protein depletion in the mitochondrial ISC machinery confers cell protection against STS-induced apoptosis.

<p>(A) HeLa cells were transfected with negative control (NC) or <i>iscu-</i>, <i>nfs1-</i>, <i>mfrn2-</i> or <i>hsc20</i>-siRNA for 6 days. Cells treated with staurosporine (STS) for 4 h were used as positive control for apoptosis induction. After staining with Hoechst 33342, cells were analyzed by epifluorescence microscopy. Scale bar: 100 μm. (B) HeLa cells were either transfected with NC or <i>iscu</i>-siRNA for 3 days or treated with STS for 4 h. Total protein extracts were analyzed by immunoblotting using antibodies against cleaved caspase 3 (C3), PARP1 and ISCU. Vinculin was used as loading control. (C) Cells were transfected with negative control (NC) or, <i>nfs1-</i>, <i>mfrn2-</i> or <i>hsc20</i>-siRNA for 6 days or STS-treated for 4 h. Total protein extracts were analyzed by immunoblotting using antibodies against cleaved caspase-3, PARP1, NFS1 and HSC20. Vinculin was used as loading control. (D) Schematic representation of the protocol used. <i>Phase 1</i>—HeLa cells were seeded the day before the transfection with <i>mfrn2-</i>, <i>iscu-</i> or NC siRNA and incubated for 6 days (144 hours) under normoxic conditions (21% O₂) with a transfection every 3 days. <i>Phase 2</i> –Cells were either untreated or treated with STS for 4 h. (E and F) Total protein extracts were analyzed by immunoblotting using antibodies against cleaved caspase-3 (C3) and ISCU. β-Actin was used as loading control. (<i>Lower panel)</i> The bar graph presents the ratio between the amounts of cleaved C3 over ß-actin determined by quantification of the immunoblot. Mean and standard deviation of n = 3 (<i>iscu</i>-siRNA) and n = 4 (<i>mfrn2</i>-siRNA) independent experiments (* p < 0.05).