Matrigel invasion assays following CRHBP expression alterations.
Real-time impedance measurement of invasiveness of the RCC-GS cell line following si-RNA-knock down of CRHBP in RCC-GS cells treated in advance with 0.125 μM (A) or 0.5 μM (B) 5-aza-2´-deoxycytidine for endogenous re-expression of CRHBP. RCC-GS cells were incubated with 25nM Target (circles) or TargetPlus control (squares) si-RNA and subsequently placed in a nutrient and growth factor deficient medium upon a membrane covered by 2,5% Matrigel layer separating the nutrient and growth factor containing second chamber. Passing through the layer and growth upon the microelectrode (cell index) was monitored against time (seconds) of measurement. Experiments were carried out in triplicate and positive and negative standard deviations of each measurement (bars) are presented. Ectopic re-expression of CRHBP following transfection of CHO control and RCC-GS target cells in western blot analysis (C) and the Matrigel invasion assay (D) using a 1.5% Matrigel layer. Single positive and negative standard deviations from quadruplicate experiments are shown for the CRHBP expression positive and negative RCC-GS cell measurements.