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. 2019 Dec 3;11(12):1923. doi: 10.3390/cancers11121923

Figure 4.

Figure 4

VGLL1 expression is regulated by the PI3K/Akt/β-catenin pathway. (a) Expression of VGLL1 mRNA after treatment with siPIK3CA or siPIK3CB. (b) NUGC3 cells incubated with inhibitors (10 µM) for 24 h. The mRNA and protein expression of VGLL1 was analyzed by RT-PCR and western blotting, respectively. (c) Effects of LY294002 on VGLL1 expression in gastric cancer cells. (d) mRNA levels of VGLL1, VGLL4, and YAP in NUGC3 cells treated with LY294002 and analyzed by RT-PCR. (e) NUGC3 cells incubated with LY294002 for 6 h at the indicated concentrations. Expression of the proteins was analyzed by western blotting. (f) Effect of AKT on VGLL1 expression assessed by western blotting. NUGC3 cells expressing wild-type (WT) AKT or constitutively active (CA) AKT were treated with LY294002. (g) Localization of β-catenin to the VGLL1 promoter. ChIP assay was performed using nuclear extracts of NUGC3 cells treated with LY294002. The ChIP-enriched DNA was subjected to PCR. (h) Effect of β-catenin expression knockdown or overexpression on VGLL1 expression in NUGC3 cells analyzed by RT-PCR and western blotting. (i) VGLL1-promoter-driven luciferase reporter activity. NUGC3 cells were treated with siRNAs and then transfected with VGLL1-luc and Renilla-luc vectors. (j) Effect of LY294002 on VGLL1 promoter activity. NUGC3 cells were co-transfected with β-catenin, p300, TCF4, LEF1, VGLL1-luc, and Renilla-luc for 24 h and then incubated with 10 µM LY294002 for 24 h. Mean ±SD of three independent experiments with triplicate measurements.* p < 0.05, ** p < 0.01, *** p < 0.005 (Student’s t-test). (k) The effects of β-catenin, p300, TCF4, and LEF1 on VGLL1 expression, assessed by western blotting.