Fig. 5: PAR-1 is a major determinant in thrombin-promoted metastasis of lung cancer and formation of VM.
a, b PAR-1 gRNA constructs targeting the PAR-1 gene via lentiviral transduction were used to construct stable A549PAR-1−/− and LLCPar-1−/− cells. An empty construct was also used as a negative control. VM channel formation in PAR-1-deficient A549 and LLC cells. Left, representative photographs of experiments. Right, quantification of the number of VM tubes. c Cell lysates were probed for phosphorylated p65, phosphorylated IκBα, and total p65 and IκBα. d The expression of snail and other EMT markers in PAR-1-deficient A549 and LLC cells was determined by western blotting analysis. e–j LLC cells infected with gRNA-PAR-1 lentivirus (Par-1−/− group) or LV-negative control (NC, vehicle group) were injected subcutaneously into the right flank of mice. e Volumes of resected tumors derived from injection of LLCPar-1−/− and LLCNC cells into mice at 5 weeks after cell injection. f Photograph of tumors resected from each group at 5 weeks after cell injection. g Number of mice from different groups with panniculus invasion, lung metastasis, liver metastasis, and colon metastasis. h Representative images of anti-CD31/PAS staining in tumor tissues. Right, the number of VM tubes per tumor tissue in the vehicle and Par-1−/− groups. i E-cadherin, N-cadherin, and Snail in resected tumors. The summarized western blot data for EMT markers are given (bottom). j DAPI and FITC-dextran (dextran inverted)-stained tumor sections from resected tumors. Right, leakiness index of resected tumors. All the results are expressed as the mean ± SD. The error bars indicate the SD. ANOVA followed by Dunnett’s test was applied for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, NS, not significant
