Figure 4

Knock-down of PECAM-1 or Pyk2 decreased cell aggregation and colony formation in soft agar. A, H460 (left) and A549 (right) cells were infected with pRetrosuper/Pyk2, pRetrosuper/PECAM-1, or scrambled (negative control) RNAi retrovirus. The cells were then selected with puromycin for 48 h. RNAi-infected cell lysates were analyzed for the level of protein expression by immunoblotting analysis using the antibodies indicated. Anti-tubulin antibody was used to show equal protein loading. B, The morphology of aggregates of H460 cells (left) and A549 (right) infected with PECAM or Pyk2 RNAi vectors. The RNAi-infected cells were selected with puromycin for 48 h. Cells were harvested cultured on 100-mm polyHEMA-coated dishes for 8 h, and photographed by phase-contrast microscope (× 40). C and D Function of PECAM-1 or Pyk2 in promoting soft agar growth of H460 (left) and A549 (right) cells. C. RNAi-infected cells were harvested and plated in soft agar grown for 2 wk at 37°C, and assayed for colony formation. D, Graphical representation of microscopy data expressed as the percentage of colonies. Data from at least three separate experiments were analyzed using Student's t test. "***": P < 0.001