Fig. 2: Measurement of luciferase reporter activity.
From: Novel start codon variant in the 5’UTR of LDLR associated with familial hypercholesterolaemia
On day 1 of growth, A549 cells in 96-well plates were transfected with 1 ng/well of phRL-CMV (expressing Renilla luciferase) plus 125 ng/well of pLDLR-UTR-Luc2P reporter plasmids containing either the wild-type LDLR 5’UTR or 5’UTR sequences with the indicated mutations. On day 2, cells were switched to cholesterol-depleting medium B in the absence or presence of 1 µg/ml 25-hydroxycholesterol (25HC). Cells were harvested and lysates analysed on day 3. Firefly luciferase activity was divided by Renilla luciferase activity. The results from three independent experiments were each normalised and then averaged. Error bars indicate SEM. A Relative firefly luciferase activity in DMEM/F12 supplemented with antibiotics (100 units per ml of penicillin and 100 µg per ml of streptomycin sulphate), 5% lipoprotein-deficient serum (LPDS), 0.5 µM lovastatin and 50 µM mevalonate (LPDS/Lov) and LPDS/Lov plus 25-hydroxycholesterol (LPDS/Lov + 25HC). B Relative firefly luciferase activity in all tested media; DMEM/F12 supplemented with antibiotics and 10% FBS (FBS), DMEM/F12 supplemented with antibiotics, 5% lipoprotein-deficient serum (LPDS), LPDS/Lov, and LDPS/Lov +25HC.
