Combined inhibition of the IGF1R and IR more effectively inhibits proliferation of Hcc193 cells than inhibition of IGF1R alone.

<p>(A) Hcc193 cells were cultured under anchorage–independent conditions in 0.1% serum in the presence of 3GF (1 nM insulin, 50 nM IGF-1, 50 nM IGF-2) alone and in combination with αIR3 (2 µg/ml), IR47-9 (2 µg/ml), αIR3 (2 µg/ml) and IR47-9 (2 µg/ml), and AZ12253801 (50 nM), as indicated. After 5 days viable cells were estimated by the fluorescence of the metabolic reduction product of Alamar blue. Each bar represents the mean fluorescence of four replicate wells ± standard deviation from a single experiment. The results shown are representative of 3 independent experiments. ***, <i>p</i><1×10⁻⁵; **, <i>p</i><0.0001; *, <i>p</i><0.01 (valid for all repeats); two-tailed unpaired <i>t</i> test. (B) Hcc193 cells were treated with αIR3 (2 µg/ml), IR47-9 (2 µg/ml), αIR3 (2 µg/ml) and IR47-9 (2 µg/ml), and AZ12253801 (50 nM) as indicated for 2 h before 10 min incubation with vehicle or 3GF. Phosphorylation of Akt on S473 and expression of IGFRβ and IRβ was determined by western blotting. An anti-α-tubulin antibody was used as a control for protein loading. All primary antibodies used here detect one prominent band at the predicted molecular weight of the protein the antibody is raised against. Scanned images of immunoblots are cropped to feature this prominent band.