Mycoplasma pcr detection kit

The MCF10CA1h and MCF10Ca1a cell lines were obtained from Dr. Fred Miller, Karmanos Cancer Institute, Detroit, MI. MCF7E (early passage MCF7 cells) were a gift from Dr. Michael Brattain, Eppley Institute, Nebraska. MDA-MB-231 LM2 cells were a gift from Dr. Joan Massague, Memorial Sloan Kettering Cancer Center, New York, NY. Additional breast cancer cell lines were all obtained from the ATCC, Manassas, VA. Cell lines not obtained directly from ATCC were authenticated by short terminal repeat (STR) analysis using 15 polymorphic markers and one gender marker (Laragen Inc, Culver City CA). All cell lines were routinely tested using Mycoplasma PCR Detection Kit (Sigma, MP0035) and shown to be mycoplasma-free. Cells were used for experiments within 2–12 passages after recovery from frozen stocks. Growth conditions are given in Supplementary Table S1. Proliferation and apoptosis were assessed using Click-iT ® imaging assays (Invitrogen). Invasion and migration were assessed by Transwell assays with or without Matrigel. Tumorsphere-forming activity was assessed in semisolid MethoCult medium (Stem Cell Technologies).

All cell lines were purchased from ATCC and maintained at 37°C and 5% CO₂ (“normoxic” condition) unless noted. The cell lines were validated using the Johns Hopkins Genetic Core Research Facility (Baltimore, MD) and stock aliquots were stored under liquid nitrogen. Testing for mycoplasma was performed using a mycoplasma PCR detection kit (Sigma-Aldrich, St Louis, MO). Py-Im polyamides were synthesized by solid-phase methods on Kaiser oxime resin (Nova Biochem, Billerica, MA) (20 (link)). The tested polyamide, HIF-PA, targets the sequence 5′-WTWCGW-3′ (W=A or T) and modulates a subset of hypoxia-induced genes, whilst the control polyamide (CO-PA) recognizes the non-HRE sequence 5′-WGGWCW-3′. Enzyme-linked immunosorbent assay (ELISA) kits specific for human VEGF was purchased from R&D Systems (Minneapolis, MN). The Hypoxyprobe-1 kit was purchased from HPI Inc (Burlington, MA). Cellular apoptosis was measured by flow cytometry using a cleaved caspase-3 kit (BD Biosciences, San Jose, CA). Small inhibitory RNA (siRNA) for HIF1α (Silencer Select siRNA ID# s6539, gene ID# 3091) and scrambled control RNA (Silencer Select negative control #1 siRNA) were purchased from Ambion (Grand Island, NY). Cells were transfected with siRNA using Lipofectamine-2000 (Life Technologies, Grand Island, NY).

Two independently-derived primary human foreskin keratinocyte lines (cultured from discarded Foreskin samples obtained at Tufts-New England Medical Center when the PI was on the faculty), and designated as 10HFK and 11HFK, were cultured in KGM medium (KGM™ Keratinocyte Growth Medium BulletKit^TM, Lonza). The TERT-immortalized keratinocyte cell line NTERT1 was kindly provided by Dr. James Rheinwald (22 (link)). The human immortalized keratinocyte cell line HaCaT (23 (link)) was maintained as described previously. HNSCC cell lines SCC1, SCC10B, SCC11 and HCC38 were obtained from Dr. Thomas Carey (University of Michigan, Ann Arbor, MI) (24 (link)) and HPV+ HNSCC cell lines UPCI:SCC90, UPCI:SCC152, and UPCI:SCC154, were obtained from Dr. S. Gollin, University of Pittsburgh Cancer Institute (25 (link)). These cell lines and 293T cells were maintained in DMEM with 10% FCS with growth factors as described in (12 ). Human cervical carcinoma cell lines HeLa (HPV18-positive), SiHa (HPV16-positive), and CaSki (HPV16-positive) were obtained from the American Type Culture Collection (Manassas, VA) and grown at 37°C in a humidified atmosphere with 5% CO₂ in α-MEM supplemented with 10% FCS and other usual supplements. All cell lines were mycoplasma negative based on testing with mycoplasma PCR Detection Kit (Sigma-Aldrich, USA).