Hush shrna plasmid panels

Smart-Pool short interfering RNA (siRNA) for various cellular targets including β-AR2 and non-target control siRNA (C-siRNA), and Dharmafect-2 transfection reagent were purchased from Dharmacon Inc. (Dharmacon-GE Life Sciences, Lafayette, CO). Recombinant chemokines and growth factors (Human SDF-1α, CXCL11, IL-8, EGF and amphiregulin) were purchased from R&D Systems (Minneapolis, MN). Full-length CXCR7 cDNA was obtained from Origene and was cloned in Ampicillin vectors for stable transfection in RWPE-1 cells. CXCR7-GFP and β-AR2 expression vectors were a gift from Dr. Kathleen Luker, University of Michigan. HuSH /shRNA Plasmid Panels for transient or stable down regulation of β-AR2 (GI326261 (1 (link)), GI326262 (2 (link)), (GI326263 (3 (link)), GI326264 (4 (link))) and scrambled shRNA (TR30013 (13 )) were purchased from Origene (Rockville, MD).

Bladder cancer cell lines (HT1376 and 5637) were obtained from American Type Culture Collection (Rockville, MD) and were used within 10 passages of cell authenticity confirmation by genetic profiling (Genetica DNA Laboratory Inc., Cincinnati, OH). 253J cells and nonmalignant bladder epithelial cells (UROTSA) were a kind gift from Dr. Colin Dinney (MD Anderson Cancer Center, Houston, TX, USA) and Dr. Donald Sens (University of North Dakota, Grand Forks, ND, USA), respectively. Fetal bovine serum (FBS) was purchased from Atlanta Biologicals (Atlanta, GA), antibiotics (gentamycin) and cell culture media were purchased from Invitrogen (Carlsbad, CA) or Sigma-Aldrich (St. Louis, MO). All cells were grown in RPMI 1640 containing 10% FBS. For stable down regulation of ARRB2, 253J cells were transfected with HuSH/shRNA Plasmid Panels (Origene). For over expression of ARRB2 cells were transfected with an ARRB2 plasmid (RC201168, Origene). The details of transfection and other routine procedures are described in Supplementary Materials and Methods.