Figure 7
Axl contributes to motility and colony formation by the 143B cell line. (a–e) The first validation strategy compared the effects of the siRNA pool with that of the individual duplexes. Knockdown efficiency was assessed by real-time PCR measurements of Axl mRNA levels (mean±s.e.m. of three PCR well replicates) (a). Effects of the siRNAs on in vitro phenotypes were assessed by measuring motility (b), colony formation (c), invasion (d) and cell growth (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 the Axl small molecule inhibitor, BGB324, with that of the siRNA pool. Cells were treated for 1 h with BGB324 or the control 1% dimethyl sulfoxide. A dose response of Axl activity inhibition by BGB324 was assessed by western blotting with a phospho-Axl antibody (f, top panel) or the total Axl antibody (f, middle panel). β-actin was assessed as a housekeeping protein (f, bottom panel). Western blot results are representative of 10 independent experiments. For expanded views of the blots with protein markers see Supplementary Figure S10. Effects of BGB324 on in vitro phenotypes were assessed by measuring motility (g), colony formation (h), invasion (i) and cell growth (j). Results are presented as mean±s.e.m. of three independent experiments.
