Figure 5

Mesothelin sliencing suppresses cell survival,proliferation and promotes apoptosis by p53-dependent and -independent pathway in pancreatic cancer cells. A, Western blot assay for p53, PUMA,bax and bcl-2 in Capan-2 cells with wt-p53. Mesothelin sliencing significantly increased the P53,PUMA and bax levels and decreased bcl-2 level. Knockdown of p53 by shRNA(3 days transfection) decreased the PUMA and bax level and increased the bcl-2 level in stable mesothelin silenced CaPan-2 cells. B, Determination of caspase-3 activity. Caspase-3 activity was determined by fluorogenic substrates. Caspase-3 activity was measured fluorometrically at 510 nm on a microplate fluorescence reader. Mesothelin sliencing significantly increased the caspase-3 activity. The activity in mock shRNA transfected cells was defined 1.^* denote p < 0.05, compared with mock shRNA controls, t test. C, Cytotoxicity assay was by MTT. .^* denote p < 0.05,^**p<0.01, compared with mesothelin shRNA groups, t test. D, Cell apoptosis was determined by FCM assay in samples treated with mesothelin shRNA or mesothelin shRNA plus PUMA shRNA. E, Cell apoptosis was determined by TUNEL assay in samples treated with mesothelin shRNA or mesothelin shRNA plus PUMA shRNA.^* denote p < 0.05, compared with combined shRNA treatment groups, t test. F, Western blot assay for p53, PUMA,bax and bcl-2 in ASPC-1 cells with mt-p53. Mesothelin sliencing significantly increased the PUMA and bax levels and decreased the bcl-2 level.