Figure 3.

Hs578T were seeded at 0.3 × 10⁶ per T25 culture flasks for 24 h after which the GM was replaced with SFM for 24 h. Cells were treated (A) with IGFBP-3 (100 ng/ml) for 30 min or (B) with IGFBP-3 (100 ng/ml) following a 30-min pre-dose with an anti-beta 1 integrin blocking antibody (200 ng/ml) or (C) a PKA inhibitor KT5720 (1 μM). Following treatment activation of Rho was assessed as described in Section “Materials and Methods.” (A–C) show Western immunoblots (of active and total Rho and are representative of experiments repeated three times) and graphs of the mean arbitrary optical density measurements from three experiments demonstrating the change in active Rho compared to the control. (D) Cells were treated with IGFBP-3 (100 ng/ml) for 30 min following a 30-min pre-dose with Y-27632 (5 μM) and shows Western immunoblots of p-MAPK and total ERK-2 and are representative of experiments repeated three times. The graph shows the mean arbitrary optical density measurements from three experiments demonstrating changes in p-MAPK corrected for ERK-2.