Fig. 4

Up-regulation of Pygo2 elevated PVT1 levels through the Wnt/β-catenin signaling pathway. a TCF4 binding sequence in the PVT1 promoter region predicted with the JASPAR website; the predicted sequence and score were shown. b Relative levels of PVT1 in PANC-1 and ASPC-1 cells treated with the Wnt/β-catenin signaling activator Wnt3a (100 ng/ml) and inhibitor XAV939 (10 μM). c Relative levels of PVT1 in PANC-1 and ASPC-1 cells treated with gemcitabine (1 μM) with or without Pygo2 siRNA or β-catenin siRNA transfection. d pGL-PVT1 promoter reporter luciferase activity in PANC-1 cells transfected with β-catenin and Pygo2 overexpression vector or Pygo2 siRNA and β-catenin siRNA treated with Wnt3a (100 ng/ml) or gemcitabine (1 μM). e Deletion mutants of the PVT1 promoter. f PANC-1 cells were subjected to ChIP assay using an antibody against TCF4, and enrichment of the PVT1 promoter fragments was shown. g and h ChIP assay in PANC-1 and ASPC-1 cells with the indicated primers and antibodies with or without Pgyo2 siRNA transfection. The Axin2 promoter was used as a positive control, and the primers to amplify its ORF regions were used as negative controls. i Schematic diagram of the generated luciferase reporter plasmids containing wild-type and mutant predicted TCF4 binding elements and relative luciferase activity after the transfection of HEK293T cells with these plasmids with or without LiCl (50 mM) treatment. j The effects of β-catenin and Pygo2 knockdown on the luciferase activity in PANC-1 and ASPC-1 cells transfected with wild-type PVT1 and PVT1 1–3 deletion mutant reporter plasmids with or without gemcitabine treatment. Data were represented as mean ± SD, *P < 0.05; **P < 0.01; ***P < 0.001