Figure 4. IGFBPs are downregulated in GR cells.

(A) Parental and A431 GR cells (10⁶ cells) were incubated for 24 hours in serum-free medium. The conditioned medium (C.M.) was concentrated by ultrafiltration, and IGF-I and IGF-II levels were determined by immunoassay as indicated in Methods. Bars represent the mean ± SD of 3 experiments. (B) Left panel: conditioned medium from 2 × 10⁶ parental and A431 GR cells was collected after 24 hours incubation and concentrated 20-fold by ultrafiltration. Medium was subjected to electrophoresis under nonreducing conditions, transferred to a nitrocellulose membrane, and incubated with ¹²⁵I–IGF-I overnight at 4°C. Signal was captured with a phosphorimager. Human serum and MCF-7 cells were used as positive controls for ¹²⁵I–IGF-I binding and IGFBP-4. Right panel: conditioned medium or 50 μg total protein from whole-cell lysates was subjected to immunoblot analysis with IGFBP-3, IGFBP-4, or Akt antibodies. (C) A431 GR cells were grown in 12-well plates in 0.5% FBS-containing medium for 72 hours with or without gefitinib (1 μM) and/or IGFBP-3 (1 μg/ml) and harvested by trypsinization. Cell numbers were determined with a Coulter Counter. Error bars represent the mean ± SD of 3 wells. Student’s t test was used for statistical comparisons. (D) A431 GR cells were treated with vehicle or gefitinib (1 μM) ± IGFBP-3 at the indicated concentrations for 6 hours. Cell lysates were prepared and analyzed with Western blots using the indicated antibodies.