Effect of CD9-Δ6 expression on platelet aggregation and metastasis induced by forced expression of Aggrus. (A) CHO/mock and CHO/Aggrus cells were transiently transfected with vectors encoding nothing (mock), CD9-WT, CD9-Δ6, or CD44. The transfected polyclonal CHO/mock+mock (pink line), CHO/Aggrus+mock (black line), CHO/Aggrus+CD9-WT (red line), CHO/Aggrus+CD9-Δ6 (green line), and CHO/Aggrus+CD44 (blue line) cells were incubated with mouse PRP. The light transmittance was monitored for 15 minutes. Blue arrow indicates time when cells are added, and red arrow indicates time when platelet aggregation begins (top left panel). Platelet aggregation-inducing activity was calculated using the following formula: volume = W2 × L / 2, where W is the short diameter and L is the long diameter. Data are means plus or minus SD of triplicate experiments (bottom panel). *P < .05; NS indicates not significant by Student t test. Expression level of each protein was confirmed by Western blot with antibodies to Aggrus, V5 tag, or β-actin (top right panels). (B) CHO/mock+mock, CHO/Aggrus+mock, CHO/Aggrus+CD9-WT, CHO/Aggrus+CD9-Δ6, and CHO/Aggrus+CD44 cells were injected intravenously into BALB/c-nu/nu mice (n = 6). At 28 days after inoculation, the number of lung surface tumor nodules was counted. Representative pictures of the lungs (left panels) and detailed numbers of the lung metastasis nodules (right panel) are shown. Data are means plus or minus SD. *P < .05; NS indicates not significant by Student t test.
