Figure 4
CerS6 is responsible for COX deficiency-induced apoptosis in response to oxidative stress. (a) Total RNA was isolated from COX10fl/fl and COX10−/− fibroblasts and transcribed into cDNA using Oligo(dT) primers. qPCR was performed using primers specific for each of the six mammalian ceramide synthases and normalized to GAPDH. Represented is mean±S.D. from three experimental replicates relative to COX10fl/fl. (b) HeLa cells were transiently transfected with human CerS6-GFP and left untreated (upper row). Transiently transfected HeLa cells (transfection efficiency was up to 50%) with human CerS6-GFP, CerS6R131A-GFP, CerS1-GFP, CerS3-GFP or empty vector (pEGFP-N3) were treated with H2O2 (200 μM) for 4 h and subsequently immunostained for activated Bax (red). Nuclei were counterstained by DAPI. (c) Cytosolic extracts were isolated from the transfected HeLa cells (as in b) after treatment with H2O2 (200 μM) for 4 h and cytochrome c and caspase-9 were detected in the cytosol of cells by WB. (d) Early apoptosis was measured in transfected HeLa cells (as in b) after treatment with H2O2 (200 μM) for 4 h by annexin V staining. (e) HeLa cells were transfected with specific siRNA targeting human CerS6 or scrambled control siRNA for 24 h (transfection efficiency was above 90%) and subjected to KCN (1 mM) for additional 24 h (left panel). 143BΔCOX cells were transfected with siRNA against human CerS6 or scrambled control siRNA (middle panel; transfection efficiency was above 80%). COX10−/− cells were transfected with siRNA against murine CerS6 or scrambled control siRNA (right panel; transfection efficiency was about 30%). Cells were then exposed to H2O2 as indicated and cell death was measured by trypan-blue exclusion after 24 h. In order to obtain significant cytotoxic response (low transfection efficiency), COX10−/− cells were treated with 300 μM H2O2 as indicated. (f) HeLa and 143BΔCOX cells (as in e) were subjected to H2O2 for 6 h and cytochrome c was detected in cytosolic extracts by WB. The error bars represent the mean±S.D. (n=3), two-tailed unpaired t-test. *P<0.05, **P <0.01, ***P<0.001
