RRIG1 regulation of gene expression and activities. All the experiments were repeated at least once with similar results. A, Western blotting. The RRIG1 sense and antisense-transfected MDA-MB-231 and MDA-MB-435 cells, respectively, were grown in G418-containing medium for 5 days and then subjected to protein extraction and western blotting analysis of gene expression. The value shown is the percentage of control, which was calculated using the formula: value of the RRIG1 sense or antisense transfected cells/value of the vector-only transfected cells after intensity of the target band of proteins from western blots was quantified by using NIH ImageJ 1.34s software and normalized to β-actin. B, Semiquantitative RT-PCR. The gene-transfected cells were grown in G418-containing medium for 5 days, and RNA was then isolated from the cells and subjected to semiquantitative RT-PCR analysis of MMP9 expression. C, RhoA activation assay. The gene-transfected cells were grown in G418-containing medium for 5 days and then subjected to the RhoA activation and western blot assays. The data were quantified using the NIH ImageJ software.