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. 2016 Jan 5;2:15039. doi: 10.1038/celldisc.2015.39

Figure 2.

Figure 2

HNit1 and hFhit repress β-catenin-mediated transcription. HEK-293 cells were transiently transfected with the respective reporter gene constructs pGL4.26BARluc/pGL4.26fuBARluc or pGL3Siamois-luc S5/S0. BAR-luc/fuBAR-luc reporter gene expression was activated by transfection of TCF-4 and β-catenin expression plasmids. The phRL-Null Renilla luciferase plasmid was used to normalize transfection efficiency. Luciferase activities were measured 24 h after transfection. For all experiments, at least three independent transfections measured in duplicate and ±s.e.m. are presented. (a) Effects of hNit1 (0.5 μg or 1 μg) and enzymatic-dead hNit1C203A (1 μg) on β-catenin transcriptional activity. (b) Siamois S5/S0 reporter gene activity is reduced after co-transfection of hNit1 in LEF-1/β-catenin but not in ΔN-LEF-VP16-transfected cells. (c) Repression of BAR-luc activity measured after transfection of hNit1 and hFhit alone, and additive effect after co-transfection of both constructs. (d) Topflash/Fopflash reporter gene activity is repressed by hNit1 in Wnt3a-stimulated cells. (e) Expression of endogenous Wnt target genes sp5 and cyclin D1 is upregulated in hNit1 knockdown MCF-7 cells. Relative mRNA expression was analysed by quantitative reverse-transcriptase PCR. Two hNit1 knockdown clones were analysed (clone 12, n=4±s.e.m.; clone 6, n=1) and normalized to values obtained from the scrambled clone. (f) Cell lysates were analysed by western blotting. Glyceraldehydes 3-phosphate dehydrogenase (GAPDH) was used as a loading control.