Fig. 5
BICC1 increases the stability of LCN2 mRNA. a HEK293T cells were transduced with either vector control or pLV-BICC1 in conjunction with the luciferase reporter pGL3-empty vector, pGL3-LCN2-promoter. Results are expressed as fold induction relative to that of corresponding cells transfected with the control vector after normalization of firefly luciferase activity to Renilla luciferase activity. b The 3’UTR sequence of human LCN2, containing one identified BICC1-binding motif. c RNA binding protein immunoprecipitation assay. RNAs were immunoprecipitated from BxPC-3 cells with an anti-BICC1 antibody and analyzed by qRT-PCR. d RNA pull-down assay. BxPC-3 cell lysates were incubated with magnetic beads bound to LCN2 3´-UTR RNA. The captured proteins were determined by Western blotting assay (L, lysate load; FT, flow-through; E, eluate). e A schematic diagram showing mutation of the BICC1-binding motif in LCN2 3’UTR from “TAAAT” to “GGGGG”. f RNA pull-down analysis of BXPC-3 cells. The lysates were incubated with wild-type or mutant LCN2 3´-UTR RNAs. g Luciferase analysis of 293T cells. 293T cells transfected with pLV-BICC1 or control vector (pLV-vector) were co-transfected with MT06-LCN2-3’UTR containing wild type (WT) or mutant (MUT) BICC1-binding site or MT06-empty vectors (MT06-Control). Forty-eight hours later, cells were subjected to dual luciferase analysis. h The indicated cells were treated with Actinomycin D for the indicated time periods and then the mRNA levels of LCN2 were detected by qRT-PCR assays, and analyzed by repeated measures analysis of variance (ANOVA). Data are shown as mean ± SD; N.S. not significant, **p < 0.01 in unpaired t-test
