Figure 3

Mutant p53 drives invasiveness and metastasis of pancreatic cancer cells in vitro and in vivo through the upregulation of cavin-1.

(A,B) Six human pancreatic cancer cell lines and one human pancreatic duct epithelial cell line (HPDE) were used to screen the level of mutant p53, cavin-1 and caveolin-1 protein and mRNA expression by western blot (WB) and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. (A) WB was performed as described²³. For the detection of p53, an antibody that can only recognize mutant, but not wild-type, p53 was used. (B) Levels of mRNA were assessed by qRT-PCR and normalized to the corresponding internal β-actin signal (ΔCT); the relative gene expression values were expressed as 2^{−ΔCT 32}. ^*P < 0.05, compared with each group. (C,D) Expression of mutant p53, cavin-1 and caveolin-1 after shRNA treatment was validated by WB and qRT-PCR. ^*,#P < 0.05, compared with the wild-type and mock group; ^**P < 0.05, compared with the shp53-mock group. (E) A Cell Counting Kit-8 was used to assay cell proliferation. No significant difference was found among groups. OD, optical density. (F,G) Cell invasiveness was assayed in Matrigel-coated Transwell Invasion Chambers. Cell cultures were incubated for 72 h and the cells that passed through the membranes were counted (at ×200 magnification). ^*P < 0.01, compared with the mock group; ^#P < 0.05, compared with the shp53-mock group. (H,I) Representative bioluminescence and hematoxylin-eosin (HE) images of tumor metastatic foci in nude mouse livers (n = 6). The morphology was detected in general and the number was counted. ^*P < 0.05, compared with the mock group; ^#P < 0.05, compared with the shp53-mock group.