Fig 2: PAR4 increases LoVo cell proliferation and survival. (A) Quantitative polymerase chain reaction analysis of PAR4 from LoVo/vector and LoVo/PAR4 cells. *P<0.05 vs. LoVo/vector cells. (B) Western blot analysis of PAR4 of whole cell lysates from LoVo/vector and LoVo/PAR4 cells. β-actin was used as a loading control. (C) Proliferation of LoVo/vector and LoVo/PAR4 cells in the presence of 10% fetal bovine serum. Results are expressed as the fold change in cell number relative to day 1 (n=3). *P<0.05. (D) Number of colonies observed from LoVo/vector and LoVo/PAR4 cells in soft agar containing 5% fetal bovine serum. *P<0.05 vs. LoVo/vector cells. PAR4, protease-activated receptor 4.

Fig 3: Effects of PAR4 on the migratory activity of colon cancer cells. (A) Migratory activity of LoVo/vector and LoVo/PAR4 cells. Left: Quantification of cells that migrated to the other side of the migration insert. Right: Migratory cells from an area randomly selected from five fields. *P<0.05 vs. LoVo/vector. (B) Migratory activity of HT-29/pGIPZ, HT-29/shPAR4#4 and HT-29/shPAR4#5cells. Left: Quantification of cells that migrated to the other side of the migration insert. Right: Migratory cells from an area randomly selected from five fields. *P<0.05 vs. HT-29/pGIPZ. PAR4, protease-activated receptor 4.

Fig 4: Overexpression of PAR4 in LoVo cells increases LoVo tumorigenesis. (A) LoVo/vector or LoVo/PAR4 cells were separately injected into nude mice subcutaneously, and the tumor volume was quantified weekly. The LoVo/PAR4 tumor increased in volume more rapidly compared with the LoVo/vector tumor, and a significant difference in tumor volume between the LoVo/vector and LoVo/PAR4 was observed from week 3 onwards. (B) Mice injected with LoVo/vector and LoVo/PAR4 cells, and (C) tumors from LoVo/vector and LoVo/PAR4 mice. (D) Average tumor weights from mice 5 weeks after injection with LoVo/vector and LoVo/PAR4 cells. The tumor volumes and weights are expressed as the mean ± standard deviation. *P<0.05 vs. LoVo/vector. PAR4, protease-activated receptor 4.

Fig 5: PAR4 is required for HT-29 cell proliferation and survival. (A) Quantitative polymerase chain reaction analysis of PAR4 expression from HT-29 cells transfected with empty vector (pGIPZ) or shPAR4 (clones #4 and #5). (B) Western blot analysis of PAR4 of whole cell lysates from HT-29/shPAR4#4 and HT-29/shPAR4#5 cells. β-actin was used as a loading control. (C) Proliferation of HT-29/pGIPZ, HT-29/shPAR4#4 or shPAR4#5 cells in the presence of 10% fetal bovine serum. Results are expressed as the fold change in cell number relative to day 1 (n=3). (D) Number of colonies observed from HT-29/pGIPZ, HT-29/shPAR4#4 or shPAR4#5 cells in soft agar containing 5% FBS. *P<0.05 vs. HT-29/pGIPZ cells. PAR4, protease-activated receptor 4; shPAR4, short hairpin RNA targeting PAR4.