Plvx ires hyg

The cnf1 gene from UPEC strains 11 was amplified by PCR and cloned into pET-28a(+) (Novagen, Madison, WI, USA). The coding sequences for RhoC, HIF1α, and HSP90α were subcloned into pCMV-Tag2B (Stratagene, La Jolla, CA, USA), pLVX-EF1α-IRES-Puro, pLVX-IRES-Hyg (Clontech, Mountain View, CA, USA) or pCDNA3.1-HA-Hygro (Invitrogen, Carlsbad, CA, USA). The constructed plasmids and primer sequences are listed in Tables S1 andS2. with Corning Matrigel Matrix (Corning Incorporated, Corning, NY, USA) for invasion assays. Cells (1 × 10 5 in 200 μL) resuspended in serum-free RPMI 1640 medium were added to the upper chamber of uncoated (for migration assays) or Matrigel-coated (for invasion assays) membranes with PBS, CNF1 (1 nmol/L), or C866S (1 nmol/L). Then, 600 μL of medium containing 20% fetal bovine serum with according PBS, CNF1 (1 nmol/L), or C866S (1 nmol/L) was added to the lower chamber. After 12 hours (migration assays) or 24 hours (invasion assays) incubation at 37 ℃ in a 5% CO 2 humidified atmosphere, the cells that adhered to the bottom surface of the inserts were fixed in 4% paraformaldehyde for 1 hour, and stained with 0.1% crystal violet (Beijing Solarbio Science & Technology Co. Ltd., Beijing, China) for 15 minutes. Finally, the filters were washed three times in PBS and images were captured under a microscope (Leica Microsystems, Wetzlar, Germany) at 200× magnification.