Figure 3. ER stress triggers cytochrome c redistribution and caspase-9-dependent cell death.

(A) WT and BAX/BAK DKO cells were treated with 10 µg/ml Tm or left untreated (NT) in regular cell culture media or in cells pre-treated with media containing 0.5% serum for 2 h. After 8 h of treatment, ΔΨ_m was determined by DIOC₈(3) staining and FACS analysis. (B) BAX/BAK DKO cells were exposed to 10 µg/ml Tm in the presence of cell culture media containing 10% serum (top panel) or pre-treated with media containing 0.5% serum (Botton panel). After 10 h, cells were stained with Mitotracker orange (red), fixed and cytochrome C (Cyt C, green) distribution visualized by indirect immuofluorescence. DNA was stained with Hoechst (blue). Images were acquired by confocal microscopy and are representative of four independent experiments. Scale bar is 25 µm. (C) Quantification of the Mandeŕs colocalization coefficient MTO/CytC (fraction of mitrotracker orange signal in co-localizing with cytochrome C signal) or CytC/MTO (fraction of the cytochrome C co-localizing mitotracker orange). Student’s T-test was used to analyze statistical significance (*: p<0.05). (D) Quantification of the percentage of cells with redistributed cytochrome C (CytC) experiments described in C (***: p<0.001). (E) Left panel: BAX/BAK DKO cells exposed to 10 µg/ml Tm for 10 h in the presence of cell culture media containing 10% (top panel) or 0.5% (bottom panel) serum were fixed and active caspase 9 (green) was evaluated by indirect immunofluorescence. Actin was monitored with Phalloidin-Rhodamine (red) and nuclei were stained with Hoechst (blue). Images are representative of four independent experiments. Scale bar is 25 µm. Right panel: Quantification of active caspase 3 fluorescence per cell (Arbitrary units: A.U.) and the percentage of active-caspase 3-positive cells. Data represents mean and standard deviation of four independent experiments performed in duplicates. Student’s T-test was used to calculate statistical significance (*: p<0.05; ***: p<0.001). (F) BAX and BAK DKO cells were stably transduced with a lentiviral vector expressing an shRNA against pro-caspase-9 mRNA (shCaspase-9) or a control shRNA against the luciferase mRNA (shLuc), and pro-caspase-9 protein levels were determined by Western blot. HSP90 levels were monitored as loading control. Then, the cells were treated with 10 µg/ml Tm in cells grown under normal cell culture media or pre-treated for 2 h with media containing 0.5% serum. Cell death was determined after 24 h by PI staining and FACS analysis. Data represents mean and standard deviation of three independent experiments performed in duplicates. Student’s T-test was used to analyze statistical significance between shLuc and shCaspase-9 cells treated with Tm (*: p<0.05; n.s.: no significant). (G) BAX and BAK DKO cells were pre-incubated in media containing 0.5% serum for 2 h. Then, the cells were exposed to 10 µg/ml Tm for 24 h in present or absence of 25 µM z-IETD-fmk (caspase-8 inhibitor). As positive control, DKO cells were exposed to 1 µg/ml Fas ligand (FasL) as positive control of working Z-IETD-FMK. The cell death was monitored by propidium iodide (PI) staining and FACS analysis. Results represent average and standard deviation of three independent experiments. Student’s T-test was used to analyze statistical significance (**: p<0.01; n.s.: no significant).