Figure 2

Tiam1 supports motility in human breast cancer cell lines. MDA-MB-231 (a, b) and MDA-MB-453 (c, d) cells were transfected with either control siRNAs (si Ctrl) or siRNAs targeting Tiam1 (si Tiam1). (a, c) Lysates were collected at 48 hours and examined with Western blot for expression of Tiam1, Rac1, and α-actinin (loading control), and with affinity precipitation assays for levels of activated Rac1 (Rac1-GTP). (b, d) Cells from replicate plates were also examined in transwell motility assays as described in Materials and Methods. Cell migration was quantified at either 6 hours (MDA-MB-231) or 48 hours (MDA-MB-453). (e) T47 D cell lines were established that stably express full-length (FL) Tiam1, an HA-epitope tagged, activated derivative of Rac1 (Rac1(61L)), or cognate vector (Ctrl), as described in Materials and Methods. Lysates were collected and examined with Western blot for expression of total Rac1, Rac1(61L) (HA), and Tiam1, and with affinity precipitation assay for levels of activated Rac1 (Rac1-GTP). The expression of α-tubulin or α-actinin was also measured as a loading control, as indicated. (f) Cells also were examined in transwell motility assays. Cell migration was quantified at 24 hours. Data shown are representative of three independently isolated stable cell lines examined in triplicate experiments (BP).