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. Author manuscript; available in PMC: 2015 Sep 26.

Published in final edited form as: Oncogene. 2014 Apr 28;34(13):1718–1728. doi: 10.1038/onc.2014.93

(a) H-ras and p53mut transformed CV-1 cells were transiently cotransfected with GFP and luciferase, ORCTL3, or caspase 2 in a 1:3 ratio and cultivated with either 100 μM BSA-conjugated oleic acid or BSA for 24 hours. Luciferase-control background was subtracted. Data are means ± SD (n=4). (b) The SCD1 inhibitor CAY10566 or equal volumes of DMSO were applied to WT as well as H-ras and p53mut transformed CV-1 cells and apoptosis was quantified after 48 hours by fluorescein diacetate (FDA) staining. (c) ORCTL3-HA was transfected into 293T cells (right, input). Cell lysates were immunoprecipitated with an α-HA antibody (left panels) that revealed multiple, probably glycosylated, forms of ORCTL3 or with an antibody against the endogenous SCD1 (middle panels). The immunoprecipitates were analysed using appropriate antibodies. (d) The desaturation index (18:1n-9/18:0) upon transfection of the indicated expression constructs into normal (top panel) and H-ras transfected (bottom panel) CV1 cells. (e) 293T cells were transfected with ORCTL3, RIP1 and ANT1 with either β-Gal or a plasmid expressing SCD1 in a 3:1 ratio. 48 hours post-transfection, apoptosis was assessed by staining the cells with DiOC₆/PI and analysing by flow cytometry. Data represent the means ± SD (n=3). Raw data were normalised to transfection efficiency estimated by GFP. (f) Endogenous SCD1 mRNA level was semi-quantitatively determined in dublicates by RT-PCR in WT-, H-ras-, and P53-mut-transformed CV-1 cells.

(a) H-ras and p53mut transformed CV-1 cells were transiently cotransfected with GFP and luciferase, ORCTL3, or caspase 2 in a 1:3 ratio and cultivated with either 100 μM BSA-conjugated oleic acid or BSA for 24 hours. Luciferase-control background was subtracted. Data are means ± SD (n=4). (b) The SCD1 inhibitor CAY10566 or equal volumes of DMSO were applied to WT as well as H-ras and p53mut transformed CV-1 cells and apoptosis was quantified after 48 hours by fluorescein diacetate (FDA) staining. (c) ORCTL3-HA was transfected into 293T cells (right, input). Cell lysates were immunoprecipitated with an α-HA antibody (left panels) that revealed multiple, probably glycosylated, forms of ORCTL3 or with an antibody against the endogenous SCD1 (middle panels). The immunoprecipitates were analysed using appropriate antibodies. (d) The desaturation index (18:1n-9/18:0) upon transfection of the indicated expression constructs into normal (top panel) and H-ras transfected (bottom panel) CV1 cells. (e) 293T cells were transfected with ORCTL3, RIP1 and ANT1 with either β-Gal or a plasmid expressing SCD1 in a 3:1 ratio. 48 hours post-transfection, apoptosis was assessed by staining the cells with DiOC₆/PI and analysing by flow cytometry. Data represent the means ± SD (n=3). Raw data were normalised to transfection efficiency estimated by GFP. (f) Endogenous SCD1 mRNA level was semi-quantitatively determined in dublicates by RT-PCR in WT-, H-ras-, and P53-mut-transformed CV-1 cells.