Fig. 2 Expressivity and application of pFila.
(a) pFila consistently produces Fluc and Rluc activities in human-, mouse- and monkey-sourced mammalian cell
lines. pFila was gradientlytransfected into the cells stated above and Fluc and Rluc luciferase levels were determined by a DLRTM Assay
(Promega). As shown in this graph, Fluc and Rluc activities were
in a linear range for all the selected points.
(b) pFila is capable of recapitulating the
interaction of miR16 and its known target CCNE1. Wild-type and mutant CCNE1 3’UTRs
were sub-cloned into pFila and co-transfected with miR16-1 mimics. Conventional
dual luciferase reporter was conducted in parallel to compare their
reproducibility. siRNA against Rluc serves as positive control. Rluc with mutant CCNE1 3’UTR was rescued in comparison with pFila-CCNE1-3’UTR-wildtype,
indicating that CCNE1 is a direct target of miR16 as previously reported (Wang et al. 2009).
(c) miR16-1 inhibitors were co-transfected
with pFila-CCNE1-3’UTR-wildtype or mutant plasmids into Hela cells by different
transfection reagents to block endogenous miR16-1. Inhibition efficacy varies
with the selected agents, where RNAiMAX achieved the most potent inhibition. It
implies that pFila is a sensitive biosensor for functional miRNA profiling.