Method: Parental and wild type c-Src breast carcinoma MDA-MB-231 cell lines were maintained with HyQ-DMEM (HyClone) plus 10% serum. Prior to plating, cells were serum-starved in HyQ-DMEM plus 0.5% BSA for 24 hours. Cells were plated on 35 mm MatTek glass bottom culture dishes coated with gelatin or fibrillar collagen type I matrix and incubated for 3 hours. Cells were allowed to invade matrices in the presence or absence of serum. Total activities of small GTPases were analyzed using ELISA-based assays (Cytoskeleton, Inc.) following the manufacturer’s protocol with slight modifications.
Result: Parental and wild type c-Src MDA-MB-231 cells were cultured on either gelatin- or collagen type I-based extracellular matrices. GTPase activity levels were determined, and the normalized values were compared. Parental and wild type c-Src clones exhibited increased RhoA and Rac1 activity upon serum stimulation. The level of activity increase in the two clones was similar. Serum stimulated RhoA and Rac1 activity on both gelatin and collagen type I matrices.
Conclusion: Serum stimulation of MDA-MB-231 cells resulted in increased total activity of RhoA and Rac1 GTPases. More studies are needed to compare Cdc42 activity in parental and wild type c-Src clones. siRNA silencing of these small GTPases will offer further insights into their roles in invadopodia formation.
Supported by NIH Intramural project DE000719-05 (K. Yamada) and Pathway to Independence Award K99CA129205 from NCI (V. Artym).
Keywords: Cell biology, Collagen, Extracellular matrix molecules and Pathology
See more of: Craniofacial Biology