FIGURE 6.

A, PANC1 cells were co-transfected with the VMP1 promoter-luciferase reporter and either a non-targeting control (NT) vector or one of two shRNAs targeting p300 (shp300 (1) and shp300 (2)). Seventy-two hours after transfection, RNA was isolated and subjected to RT-PCR to investigate mRNA levels of VMP1, p300, and GAPDH. B, lysates from PANC1 cells transfected with two different shRNA targeting p300 were isolated, and the relative changes in luciferase activity were analyzed. C, relative luciferase activity in PANC1 cells co-transfected with the VMP1 promoter-luciferase reporter and NT vector control or p300 shRNA (shp300 (Pool)), in the presence or absence of a GLI3-expressing vector. D, PANC1 cells were transfected with the VMP1 promoter-luciferase reporter construct and a p300-expressing construct or control vector, in the presence or absence of GLI3 expression. After 24 h, proteins were isolated and used to determine VMP1 reporter activity by luciferase assay. Bars represent average levels in each group ± S.E. E, PANC1 protein lysates were immunoprecipitated (IP) with the antibodies indicated, and Western blot (WB) was performed to demonstrate coimmunoprecipitation between GLI3 and p300. F, p300 and GLI3 ChIP assay in PANC1 cells transfected with non-targeting control (NT) and a GLI3 shRNA construct.