Primary human foreskin fibroblasts (HFFs), immortalized human
fibroblasts (BJ5Ta and BR5, ATCC), human adenocarcinoma line MDA-MB-231, primary
human osteoblasts (NhOst, Lonza), and HEK 293FT cells were cultured in phenol
red-free DMEM (Hyclone) supplemented with 10% fetal bovine serum (Hyclone), 100
U/ml penicillin, 100 μg/ml streptomycin (Invitrogen), and 2 mM
L-glutamine (Invitrogen) at 37°C in 10% CO2 in a humidified
incubator. Human umbilical vein endothelial cells (HUVECs) and human aortic
smooth muscle cells (AOSMCs, Lonza) were cultured in phenol red-free DMEM
(Hyclone) supplemented with 5% fetal bovine serum (Hyclone), insulin, hFGF, and
hEGF (Lonza, SMGM-2 BulletKit) at 37°C in 5% CO2. The
following reagents were used in this study: rhodamine- and Alexa488-phallodin
(Invitrogen), cell-permeable C3 transferase (Cytoskeleton), blebbistatin and
okadaic acid (EMD), and GFP-TRAP GFP-binding protein (Chromotek). GFP-RhoQ63L
was transfected into cells with the Nucleofector system (Lonza) using the NDHF
kit (Lonza) according to the manufacturer's instructions. Equal concentrations
of the DMSO vehicle were used as controls for drug studies.
fibroblasts (BJ5Ta and BR5, ATCC), human adenocarcinoma line MDA-MB-231, primary
human osteoblasts (NhOst, Lonza), and HEK 293FT cells were cultured in phenol
red-free DMEM (Hyclone) supplemented with 10% fetal bovine serum (Hyclone), 100
U/ml penicillin, 100 μg/ml streptomycin (Invitrogen), and 2 mM
L-glutamine (Invitrogen) at 37°C in 10% CO2 in a humidified
incubator. Human umbilical vein endothelial cells (HUVECs) and human aortic
smooth muscle cells (AOSMCs, Lonza) were cultured in phenol red-free DMEM
(Hyclone) supplemented with 5% fetal bovine serum (Hyclone), insulin, hFGF, and
hEGF (Lonza, SMGM-2 BulletKit) at 37°C in 5% CO2. The
following reagents were used in this study: rhodamine- and Alexa488-phallodin
(Invitrogen), cell-permeable C3 transferase (Cytoskeleton), blebbistatin and
okadaic acid (EMD), and GFP-TRAP GFP-binding protein (Chromotek). GFP-RhoQ63L
was transfected into cells with the Nucleofector system (Lonza) using the NDHF
kit (Lonza) according to the manufacturer's instructions. Equal concentrations
of the DMSO vehicle were used as controls for drug studies.