Fig. 4.

SCCA1 promotes mitochondrion-independent apoptosis in response to ER stress. (A) BMK wild-type cells, as well as bax^−/− bak^−/− parental cells or those expressing SCCA1 or SCCA1-F352A were treated for the indicated amount of time with tunicamycin (0.5 μg/ml). Subcellular fractionation was performed, and the fractions were probed with indicated antibodies. S-100, soluble portion of the cell homogenate following centrifugation at 100,000 × g. (B) BMK wild-type, as well as bax^−/− bak^−/− parental and SCCA1-expressing cells were treated with tunicamycin for 24 h. Subcellular localization of cytochrome c (green) was visualized by immunofluorescence. DAPI (blue) was used to stain the nucleus. (C) bax^−/− bak^−/− BMK parental and SCCA1-expressing cells were treated with tunicamycin. Apoptotic nuclear morphology was visualized by DAPI staining. Quantification of fragmented nuclei was determined by performing three independent counts of 50 cells. The mean ± standard error of the mean is shown. (D) Cell lysates from bax^−/− bak^−/− BMK parental cells and SCCA1-expressing cells, treated with tunicamycin, were probed with indicated antibodies. Wild-type BMK cells were used as a positive control for apoptosis induction. (E) bax^−/− bak^−/− BMK parental and SCCA1-expressing cells were treated with tunicamycin (0.5 μg/ml) for 16 h. Caspase-8 activity was detected using the fluorogenic caspase-8 substrate FITC-IETD-FMK. (F and G) Parental and SCCA1-expressing cells containing either an shRNA control (shControl) or shSCCA1 were treated with tunicamycin. Cell death was measured by PI exclusion (F), and immunoblotting was performed to observe caspase-8 and PARP cleavage (G). (H) MCF10A parental cells, SCCA1-expressing cells, and SCCA1-expressing cells containing a Tet-inducible shSCCA1 either left untreated or treated with doxycycline for 3 days were treated with tunicamycin (5.0 μg/ml). The amount of cleaved caspase-8 was determined by densitometric analysis of cleaved caspase-8 against tubulin.