Figure 5. PinX1 suppress ccRCC metastasis in vivo.

a. left panel, Western blotting of PinX1 from PinX1^OE-786-O cell lines, PinX1^KD-786-O cell lines and Ctrl-786-O cell lines selected with puromycin for 3 weeks after lentivirus infection. Right panel, PinX1 expression levels were not changed in PinX1^OE, PinX1^KD and Ctrl 786-O cell lines without puromycin selection for 2 months. b. Representative image of lung with metastatic nodules (left panel) and H&E staining sections of lung (right panel) 2 months after injection of PinX1^KD 786-O cell lines in BALB/c nude mouse through tail vein. Arrows indicate metastatic nodules. c. top panel, Representative images of 10% buffered formalin fixed lungs with metastatic nodules 2 months after respective injection of Ctrl, PinX1^OE and PinX1^KD 786-O cell lines. Arrows indicate metastatic nodules. Bottom panel, the number of lung metastatic nodules was counted under a dissecting microscope. A statistically dramatic increase in the number of the lung metastases was seen in PinX1^KD group, compared with the PinX1^OE group and these two groups also had significant diversity compared with Ctrl group respectively. Data are displayed with means ± SD from 12 mice in each group. **, P < 0.01; ***, P < 0.001. d. Immunostaining of PinX1, MMP-2, p65 and TIMP-2 in metastatic nodules of PinX1^OE, PinX1^KD and Ctrl 786-O groups. MMP-2 and p65 expression in PinX1^OE group were much lower compared with PinX1^KD group and Ctrl group but TIMP-2 expression was not change in every group.