FIGURE 4.

The role of β3-integrin in IGF signaling in anchorage-independent conditions. a, secretion of WT IGF1 or R36E/R37E in CHO and β3-CHO cells. Cells were cultured with DMEM with 10% FCS. The culture medium was concentrated five times and analyzed by Western blotting using anti-6-His antibodies (IGF1 has an N-terminal 6-His tag). R, R36E/R37E. b and c, cell survival of the transfectants in polyHEMA-coated plates. The transfected cells were plated in polyHEMA-coated plates, serum-starved in DMEM, and cultured for 48 h. Cell survival was measured by MTS assays. The data are shown as mean ± S.E. (n = 6). Statistical analysis was performed using ANOVA and Tukey's multiple comparison test. d and e, colony formation of the transfectants in three-dimensional culture (in soft agar). The transfected cells were cultured in soft agar for 3 weeks, and the number of colonies was counted from digital images using ImageJ software. Statistical differences were tested using ANOVA and Tukey's multiple comparison test (n = 10).