Figure 4. hnRNPA2 acts on the 3′-UTR of CTNNB1 mRNA resulting in an increased β-catenin protein expression. (A) UCSC Genome Browser showing transcript details for CTNNB1. Custom track showing locations of hnRNPA2 binding sites in 293T cells.¹⁴ (B) HEK293 cells were transfected with either pMiR-report-β-catenin-3′-UTR or pMiR-report-empty vector and pRL-null reporters, and HA-tagged wild-type (WT) hnRNPA2 or hnRNPA2-ΔRGG (50 ng, 100 ng, or 200 ng) or empty vector (EV). After 48 h cells were harvested for luciferase activity analysis, and data normalized to activity of pRL-null (DNA transfection control) vector as well as pMiR-report-empty (luciferase expression control) vector. Data from at least three independent experiments with at least three technical replicates were used to calculate the means ± SE (*P = 0.001, **P = 0.02, ***P = 0.009, ^#P = 0.002, ^##P = 0.01). (C) HEK293 cells transfected with 2 µg of plasmid DNA vectors encoding HA-tagged WT hnRNPA2 or hnRNPA2-ΔRGG. qRT-PCR was performed on cDNAs from two independent experiments, and levels of CTNNB1 transcript expression were normalized to a mean of CASC3 and ACTB levels to obtain mean relative normalized fold change ± SE (*P = 0.006, **P = 0.04). (D) HEK293 (left panel) PC3 (right panel) cells transfected with 2 µg of plasmid DNA vectors encoding HA-tagged WT hnRNPA2 or hnRNPA2-ΔRGG using antibodies to HA tag and β-catenin. Western analysis images shown are representative of three independent experiments, from which densitometric band quantitation was performed to calculate the mean relative normalized fold change in total β-catenin protein expression (shown in brackets). (E) PC3 cells were transfected with 2 µg of plasmid DNA vectors encoding HA-tagged WT hnRNPA2 or hnRNPA2-ΔRGG and images captured by confocal laser scanning microscopy using indirect immunofluorescence and antibody to β-catenin (green) and a DAPI nuclear counterstain (blue). Indirect immunofluorescence images shown are representative of two fields of view, from which densitometric fluorescence quantitation was performed to calculate means relative normalized fold-change in β-catenin nuclear protein expression ± SD (All P values shown are for comparisons with control conditions).