Treatment protocol 1: Beas-2B cells were seeded at 3X105 into 25 cm2 polystyrene tissue culture flasks. The flasks then received media containing 10 µM NaVO3 and the cells were cultured for 4 weeks. Control cells received distilled water in place of NaVO3. Control parental cells did not recieve distilled water. Every 3-4 days, the cells were trypsinized, counted, and re-seeded into fresh 25 cm2 flasks at a density of 3X105 live cells/flask, and given fresh media with 10 µM NaVO3. A subset of cells were only treated for 24 hours (acute exposure). Soft Agar Assay: Warm (37oC, 2 mL/well) base agar solution (0.5% [w/v] agar [#214530, BD Biosciences, San Diego, CA] in 1X DMEM complete culture media) was poured into each well of 6-well untreated polystyrene plates. The base layer was allowed to solidify at 4oC for 2 hours. Then 2 mL of a warm (37oC) top agar solution consisting of 5,000 cells in 0.35% agar + 1X DMEM complete tissue culture media was added over the base layer. Duplicate wells were used for each treatment and control cells. The 6-well plates were then incubated at 37oC in the tissue culture incubator for 3 weeks; 0.5 mL of fresh media was added once per week without disturbing the cells. After 3 weeks, the colonies were either stained overnight with INT/BCIP solution (#11-681-460-001, Roche, New York, NY) in 0.1 M Tris/0.05 M MgCl2/0.1 M NaCl to visualize (and subsequently permit a count of) all colonies, or colonies were instead extracted from the agar. Each extracted colony was trypsinized in a 96-well plate in order to break up the colony, and then transferred into a 24-well plate to begin expansion of the individual transformed and control clones. Control parental cells were not grown in soft agar. Treatment protocol 2: Beas-2B cells were seeded at 3X105 into 25 cm2 polystyrene tissue culture flasks. The flasks then received media containing 10 µM NaVO3 and the cells were cultured for 4 weeks. Control cells received distilled water in place of NaVO3. Every 3-4 days, the cells were trypsinized, counted, and re-seeded into fresh 25 cm2 flasks at a density of 3X105 live cells/flask, and given fresh media with the appropriate concentration of NaVO3. A subset of these cells were cultured in media with 10 µM NaVO3 for 1 extra week, totalling 5 weeks of exposure, and the second set was cultured in the presence of NaVO3 for 4 weeks and then recovered in the absence of NaVO3 for 1 week.
Growth protocol
Immortalized human bronchial epithelial cells (Beas-2B; #CRL-9609, ATCC, Manassas, VA) were cultured in 1X Dulbecco’s Modified Eagle Medium (DMEM; ordered in-house from NYU) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA) and 100ug/mL Pen Strep (GIBCO, Grand Island, NY) (“complete culture media”). The cells were maintained in 25 cm2 polystyrene tissue culture flasks in an incubator at 37oC with 5% CO2 and 100% humidity. The media was changed every 2 days, and the cells were passaged every 4 days using 0.05% trypsin-EDTA (GIBCO).
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using Trizol (Invitrogen). The isolated T.RNA was subjected to a colum purification using Rneasy Plus Micro Kit (Qiagen)
Label
biotin
Label protocol
Single-stranded cDNA was generated from the amplified cRNA with the WT cDNA Synthesis Kit (Affymetrix) and then fragmented and labeled with the WT Terminal Labeling Kit (Affymetrix).
Hybridization protocol
Samples were hybridized with Affymetrix Human Gene 1.0 ST Arrays and scanned at the NYU Cancer Institute Genomics Facility.
Scan protocol
Array scanning was performed according to the manufacturer's instruction (Affymetrix)
Description
treatment protocol 1
Data processing
Data were processed using R. Data were imported and normalized using SCAN.UPC with a custum library provided by BrainArray [HuGene10stv1_Hs_ENTREZG_15.1.0]. Data presented contain EntrezIDs - expression set was filtered to remove negative expression values. EntrezID mapped to gene symbol using R package org.Hs.eg. Data were analyzed for significance using LIMMA with FDR correction.
