Substrate-Bound Insulin-Like Growth Factor (IGF)-I-IGF Binding Protein-Vitronectin-Stimulated Breast Cell Migration Is Enhanced by Coactivation of the Phosphatidylinositide 3-Kinase/AKT Pathway by v-Integrins and the IGF-I Receptor

Supplemental Data

Supplemental Data

Files in this Data Supplement:

• Supplementary Figure 1 - Supplementary Figure 1: Effect of substrate-bound IGF-I:IGFBP:VN complexes on cellular migration. T47D breast carcinoma cells were seeded onto Transwells^TM that had been coated with VN and IGF-I pre-bound in the presence or absence of IGFBP-3 and allowed to migrate for 5 hours. The number of cells which transverse the membrane in response to each treatment was expressed as a percentage of those that migrated on VN only. Data is from one representative experiment with treatments tested in triplicate wells. The asterisk indicates treatments which significantly increased migration above VN only wells (p<0.05). IGF-I:IGFBP-3:VN complexes were also observed to increase migration above the individual components of the complex (†)(p<0.05). Error bars indicate SEM.
• Supplementary Figure 2 - Supplementary Figure 2: Effect of substrate-bound IGF-I:IGFBP:VN complexes on cellular attachment. MCF-7 (A) and MCF-10A cells (B) were grown for 18 hours in the presence of 2 μCi/ml of ³H-leucine (Amersham Biosciences, Castle Hill, NSW, Australia), thus allowing the cells to be labelled via the incorporation of ³H-leucine into newly synthesized protein. Following serum starvation for 4 hours, cells were harvested by trypsinisation and 1 x 10⁵ cells were seeded into pre-coated wells of 24-well plates and incubated for 4 hours at 37ºC + 5% CO2 to allow cell attachment. After washing and trichloroacetic acid precipitation of cellular protein, cellular attachment was determined by sub-sampling solubilized protein precipitate for β-scintillation counting. The data are expressed as the average percent stimulation of control wells containing VN only. The asterisk indicates treatments which significantly increased attachment above VN only wells (p<0.05). The data is pooled from three experiments with treatments tested in triplicate wells. Error bars indicate SEM.
• Supplementary Figure 3 - Supplementary Figure 3: Involvement of VN-binding integrins and the IGF-IR in response to VN, IGF-I, IGFBP-3 and IGFBP-5-stimulated migration. Migration of MCF-7 (A) and MCF-10A cells (B) was determined in the presence of monoclonal anti-integrin blocking antibodies against αv (1:10), αvβ5 (25 μg/ml), αvβ6 (25 μg/ml), β1 (10 μg/ml) and the IGF-IR (10 μg/ml) or control mouse IgG (25 μg/ml). Cells were allowed to migrate for 5 hours after which the migration in the presence of IgG control antibodies was taken as 100%. The asterisk indicates antibody treatments which significantly inhibited migration when compared to the response in the presence of control IgG antibodies (p<0.05). The data is pooled from three experiments with treatments tested in duplicate wells. Transwells^TM were pre-coated with IGF-I (30 ng/ml), IGFBP-3/-5 (90 ng/ml) and VN (1 μg/ml). Error bars indicate SEM.