Figure 6

AUY922 and specific knockdown of FN1 both suppress prostate cancer cell invasion. (A) Cells were cultured with either AUY922 (25, 50 nM), or vehicle (DMSO) for 48 h before harvesting for the invasion assay. (B) PC-3 cells were transfected with 2 independent siRNAs against FN1 (Si-1, Si-2), or non-targeting siRNA as a negative control for 48 h before harvesting for the invasion assay. (C) PC-3 cells were treated with pUR4 (500 nM) or the control peptide III-11C (500 nM) for 48 h before harvesting for the invasion assay. 3D invasion assays were conducted in PC-3 cells using 24-well Falcon BioCoat Matrigel invasion chambers. Treated or siRNA-transfected cells were seeded into the upper chamber of the Transwell at a density of 1x 10⁵ cells/well in serum-free medium. The bottom chamber was filled with RPMI-1640 medium containing 5% FBS and inserts incubated at 37 °C for 48 h. Migrated cells remaining on the bottom surface were fixed in formalin and stained with crystal violet. Five random fields for each membrane were photographed using the Axio Scope A1 Fluorescent Microscope (Zeiss), and the number of migrated cells was counted manually and presented as percent of control cells ± SEM.