Fig. 3: Measurement of luciferase and HiBiT reporter activity.
From: Novel start codon variant in the 5’UTR of LDLR associated with familial hypercholesterolaemia
A549 cells were grown as in Fig. 2 and transfected with 10 ng/well of pCATlac (expressing β-galactosidase) plus 125 ng/well of pLDLR-UTR-HiBiT-Luc2P reporter plasmids. On day 2, cells were switched to cholesterol-depleting medium B in the absence or presence of 1 µg/ml 25-hydroxycholesterol (25HC). Enzyme activities were analysed on day 3. NanoLuc (indicating HiBiT expression) was corrected for β-galactosidase. Error bars indicate SD (n = 3). Sequences that differ between the four reporters are highlighted in red. Plasmid (b) differs from wild-type plasmid (a) by a c.−35C > G mutation in the LDLR 5’UTR; the mutation introduces an upstream AUG that is out of frame with the HiBiT-Luc2P CDS. Plasmid (c) differs from plasmid (b) by the insertion of two G nucleotides following the LDLR Kozak sequence, which brings HiBiT-Luc2P in frame with the upstream AUG. Plasmid (d) differs from plasmid (c) in that sequences surrounding the upstream AUG were modified to match the core Kozak consensus sequence.
