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. 2017 Apr 6;18:73–82. doi: 10.1016/j.ebiom.2017.04.003

Fig. 3.

Fig. 3

Mutant PRPK (Ser250Ala) decreases cell migration and invasion of HCT116 colon cancer cells and the Ser250 site of PRPK are important for the function of PRPK in a human colon cancer HCT116 cell xenograft metastasis model. (a, upper left panel) HCT116 cells were transfected with pcDNA3-V5-vector (Mock), pcDNA3-V5-PRPK (WtPRPK) or pcDNA3-V5-PRPK-S250A mutant (MutPRPK) and selected with G418 (200 μM/ml) for 5 days. Expression of PRPK in Mock, WtPRPK, or MutPRPK stable cell lines was detected with an antibody against the V5 tag and β-actin was used to verify equal protein loading. (a, upper right panel) Phosphorylation of PRPK was detected in WtPRPK or MutPRPK stable cell lines after treatment by EGF (20 μg/ml, 15 min). (a, lower panel) Cells stably expressing Mock, Wt-PRPK or Mut-PRPK were subjected to a wound closure assay. Photographs were taken immediately after scratching and 18 h later using a microscope and a Casio Digital Camera (EX-Z1000 with 10.1 megapixels). Experiments were repeated twice. (b) Migration of cells stably expressing Mock, WtPRPK or MutPRPK after 18 h was determined by staining with DAPI and visualized by confocal microscope (NIKON C1si Confocal Spectral Imaging System; magnification 60 ×). Invading cells were counted from five random fields in two membranes and experiments were repeated twice. Asterisks (**) indicate a significant (p < 0.002) decreased migration of MutPRPK cells compared with WtPRPK cells. (c) Quantification of the area of liver metastasis using the ImagePro Plus 6.2 software program. The area of metastasis in livers of mice (6 mice/group) injected with WtPRPK HCT116 cells is significantly increased (***, p = 0.036) compared to livers from mice injected with Mock HCT116 cells. The metastasis (arrows) in the liver and tumor in the spleen (circles) are shown (one representative mouse per group) at 7 days after injection of cells. (d) The area of metastasis in livers from mice injected with MutPRPK HCT116 cells is significantly decreased (***, p = 0.011) compared with the area of metastasis in livers of mice injected with WtPRPK HCT116 cells. Metastasis (arrows) in the liver is shown (one representative mouse per group) at 7 days after injection of cells. (e and f) H&E staining of non-metastatic and metastatic liver tissue (4 μm sections) is shown (100 × magnification).