To stably express Pak4 in SKOV-3, cells were transfected with Flag-tagged wt Pak4, ca Pak4 (445N/474E), kinase-dead Pak4 (M350), or the control vector p3XFLAG-CMV-10 (11) using Lipofectamine 2000 (Invitrogen) and then selected with G418 (800 μg/mL) (5). For drug or siRNAs treatment, Pak4 overexpressing cells were plated 6 or 24 h before treating with the c-Src inhibitor PP1 (20 μM), the two MEK-1 inhibitors U0126 (20 μM) and PD 98059 (50 μM), the two EGFR inhibitors CL387, 785 (1 μM) and PD153035 (2 μM), vehicle (DMSO), or siRNAs (100 nM; Ambion) of c-Src, MEK-1, MMP2, or control. All inhibitors were purchased from Calbiochem except PP1 (Biomol). After 48 h (for PP1, U0126, PD98059, and siRNAs) or 12 d (for CL and PD153035 with change of medium and drugs in every 3 d), cells were harvested for real-time PCR and/or immunoblot analyses. To generate the Pak4 fusion protein with GAL4-DNA binding domain construct, wt Pak4 was amplified and subcloned in-frame into the vector pCMV-BD (Stratagene). To generate Pak4 NLS mutants, a QuikChange Kit (Stratagene) was used, and the lysine residues in the four NLSs were mutated to alanines using pCMV-BD wt Pak4 as template. Primers used are described in SI Materials and Methods. Nuclear localization of wt and NLS mutants of Pak4 were determined by immunoblotting. The constructs or the control vector were transiently transfected into SKOV-3 cells along with pFR-Luc reporter plasmid (Stratagene). After 24 h, luciferase assay was performed using Promega luciferase assay system. The transcription activation domain of NF-κB fused with GAL4 was used as positive control. To transiently silence Pak4 in OVCAR-3 and OVCA420, 100 nM each of siGENOME Smart-pool for Pak4 and siControl nontargeting siRNA pool (Dharmacon) was used. Cells were plated for migration and invasion assays 48 h after transfection. To stably silence Pak4, cells were transfected with a set of shRNA constructs against human Pak4, pRS-shPak4 (Origene), and then selected with puromycin (1.5 μg/mL) (5, 38). The pRS vector was used as controls. Full paper
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