Fig. 5

SCD inhibition alters cellular cardiolipin composition leading to cytochrome C release and sensitisation towards apoptosis. a DU145 cells were treated with different concentrations of SCD inhibitor (SCDi II) and proliferation was determined by BrdU labelling. b Cells treated as in a were used to determine apoptosis using Annexin V staining. Data represent mean ± SEM of three independent biological replicates. Statistical comparisons were performed using Student t test (*p ≤ 0.05). c Quantitative lipid profiling of cardiolipin (CL) content of DU145 cells grown for 48 h in medium containing full (FS) or low (LS) serum using LC-MS/MS. Concentrations of mono-unsaturated (≤4 double bonds) and poly-unsaturated (>4 double bonds) CL species are displayed. Data represent the mean ± SEM of three independent biological replicates. Statistical comparisons were performed using Student t test (***p ≤ 0.001). d DU145 cells were treated with SCD inhibitors (100 nM) or solvent (DMSO) for 48 h in medium containing low serum or medium supplemented with BSA or BSA-coupled oleic acid (BSA-Oleate). Quantitative lipid profiling of CL content was determined using LC-MS/MS. Concentrations of mono- and poly-unsaturated CL species are displayed. Data represent the mean ± SEM of three independent biological replicates. Statistical comparisons were performed using Student t tests (*p ≤ 0.05). e DU145 cells were treated with SCD inhibitor (SCDi I) or solvent (DMSO) for 48 h in medium containing low serum or medium supplemented with BSA or BSA-oleate. Cells were lysed by digitonin, and the presence of cytoplasmic cytochrome C was determined. UV treatment was used as positive control. Vinculin is shown as loading control. Levels of phosphorylated Akt (S473) and total Akt were detected in total lysates. f DU145 cells were treated as in e but 3 μM of Akt inhibitor was added prior to addition of BSA-oleate. Cytoplasmic cytochrome C was detected in digitonin lysates. g DU145 cells were treated with SCD inhibitor (SCDi I) or solvent (DMSO) in medium containing full (FS) or low (LS) serum or medium supplemented with BSA or BSA-oleate. The presence of full-length and cleaved (cl.) PARP was determined. Actin is shown as loading control. h DU145 cells were transfected with siRNA targeting SCD (siSCD) or non-targeting controls (siCtrl) and cultured as in e. The presence of full-length and cleaved (cl.) PARP was determined. Actin is shown as loading control. i DU145 or MDA-MB-468 cells were treated with SCD inhibitor (100 nM) for 48 h, and oxygen consumption rate (OCR) before and after addition of oligomycin, FCCP and rotenone was determined using a Seahorse Bioanalyzer. j–m DU145 cells were treated with the indicated doses of SCD inhibitor (SCDi II) either alone or in combination with different doses of metformin (Metf, j), rotenone (RN, k), paclitaxel (PTX, l) or staurosporin (STP, m) for 72 h in medium containing low serum. Cell viability was determined by crystal violet staining. Data represent the mean ± SEM of three independent biological replicates. Statistical comparisons were performed using Student t tests (*p ≤ 0.05, **p ≤ 0.01 compared to SCDi alone; #p ≤ 0.05, ##p ≤ 0.01 compared to no SCDi)