Apoptotic and necrotic β-cells and INS-1E cells were counted in fluorescence microscopy following staining with the DNA-binding dyes propidium iodide (5 μg/ml) and Hoechst 33342 (10 μg/ml) (11 (link)). The percentage human islet cell death was determined as described (12 (link)). The apoptosis index was calculated as [(% apoptosis in ER stressor–treated cells under indicated conditions transfected with negative or target siRNA − % apoptosis in ER stressor–treated negative siRNA-transfected cells)/(100% − % apoptosis in ER stressor–treated negative siRNA-transfected cells)] × 100. Apoptosis was also assessed by the Cell Death Detection ELISAplus kit (Roche Diagnostics, Mannheim, Germany) (10 (link)). Forskolin did not modify INS-1E cell proliferation under the present experimental conditions (assessed by BrdU incorporation, data not shown).
To study cytochrome C release, cells were prepared as described in the online appendix supplementary Methods. After total protein quantification (Bio-Rad Laboratories, Munchen, Germany), equal amounts of protein were blotted using tubulin as cytoplasmic and apoptosis-inducing factor (AIF) as mitochondrial control.
Caspase 3 and 7 activation was measured with the Caspase-Glo 3/7 assay (Promega, Madison, WI) and caspase 12 activation with the CaspGlow Caspase-12 staining kit (Biovision, Mountain View, CA) (12 (link)).
To study cytochrome C release, cells were prepared as described in the online appendix supplementary Methods. After total protein quantification (Bio-Rad Laboratories, Munchen, Germany), equal amounts of protein were blotted using tubulin as cytoplasmic and apoptosis-inducing factor (AIF) as mitochondrial control.
Caspase 3 and 7 activation was measured with the Caspase-Glo 3/7 assay (Promega, Madison, WI) and caspase 12 activation with the CaspGlow Caspase-12 staining kit (Biovision, Mountain View, CA) (12 (link)).
