Figure 3
IGF-1R contributes to invasion, cell growth, motility and colony formation by the LM7 cell line. (a–e) The first validation strategy compared the effects of the siRNA pool to that of the individual duplexes. Knockdown efficiency was assessed by real-time PCR measurements of IGF-1R mRNA levels (mean±s.e.m. of three PCR well replicates) (a). Effects of the siRNAs on in vitro phenotypes were assessed by measuring invasion (b), cell growth (c), motility (d) and colony formation (e). Results are presented as mean±s.e.m. of the number of replicates indicated in the Materials and methods for each assay. (f–j) The second validation strategy compared the effects of an IGF-1R neutralizing antibody to that of the IGF-1R siRNA pool. Cells were treated for 4 h with the neutralizing antibody. Effects of the antibody and the siRNA pool on IGF-1R activity were assessed by immunoprecipitation with a total IGF-1Rβ antibody followed by western blotting with a phospho-IGF-1R/IR antibody (f, top panel) or the total IGF-1Rβ antibody (f, middle panel). β-actin was assessed in total cell lysates as a housekeeping protein (f, bottom panel). Western blot results are representative of three independent experiments. For expanded views of the blots with protein markers see Supplementary Figure S10. Effects of the neutralizing antibody and the siRNA pool on in vitro phenotypes were assessed by measuring invasion (g), cell growth (h), motility (i) and colony formation (j). Results are presented as mean±s.e.m. of three independent experiments.
