Golgi reorientation in MDA-MB-231 cells is supported by Tiam1 and Rac1. MDA-MB-231 (a, b, e, f) and MDA-MB-453 (c, d) cells were examined for serum-dependent Golgi reorientation in a wound-healing assay, as described in Materials and Methods. (a, c) Confluent cells were transfected with either 200 nM control siRNA (si Ctrl) or Tiam1 siRNA (si Tiam1) overnight, serum starved for 12 hours, and then wounded. Alternatively, nontransfected cells were serum starved overnight in the presence or absence of 50 μM Rac1 inhibitor (+NSC23766) and were then wounded (e). After wounding, cells were cultured in 10% serum for 90 minutes (MDA-MB-231) or 210 minutes (MDA-MB-453), and then fixed and stained for phalloidin (green), GM130 (red), and DAPI (blue). Golgi orientation was then scored. Cells treated with the Rac inhibitor were maintained on the inhibitor throughout the course of the experiment. a, c, and e show the results of a typical reorientation experiment, with plus signs indicating cells with Golgi oriented toward the wound, whereas minus signs indicate no orientation. b, d, and f show quantification of three independent experiments. (g) Schematic showing the method of scoring of the reorientation experiments.