Fig. 6

Restoration of RhoBTB1 expression restores normal Golgi morphology to T47D cells and inhibits invasion. a T47D cells were transduced with a lentiviral RhoBTB1 construct to restore RhoBTB1 expression. Expression of RhoBTB1 and RhoBTB2 was measured by RT-PCR. Transduction with RhoBTB1 restored expression to near normal levels, but did not affect expression of RhoBTB2. Data are means ± SEM (n = 2). b Cells were fixed and stained for the Golgi marker giantin. Restoration of RhoBTB1 expression caused a significant reduction in Golgi fragmentation. Data are means ± SEM (n = 3). c shows representative images of control T47D cells, and the T47D line expressing RhoBTB1. Golgi are stained in green; nuclei in blue. Bar = 10 μm. d T47D cells were grown to confluence and cell migration was initiated by scratching the coverslip to remove a strip of cells. Cells were fixed at 1 h and stained for Golgi (giantin, green). The dashed line indicates the cell front. Quantification of Golgi polarization showed that T47D cells had little ability to polarize (25% polarization corresponds to random orientation in this assay); whereas restoration of RhoBTB1 supported robust polarization. Data are means ± SEM (n = 3). e Migration was measured in the same assay over 14 h. Data are means ± SEM (n = 3). Restoration of RhoBTB1 expression had no effect on T47D cell migration in 2D. f T47D invasion through 3D extracellular matrix was measured in Matrigel invasion chambers over 24 h. Restoration of RhoBTB1 expression markedly reduced invasive capacity. Data are means ± SEM (n = 3). *P <0.05; **P <0.01, ***P <0.001, ****P <0.0001