Refer to Whitehead, R. H., VanEeden, P. E., Noble, M. D., Ataliotis, P. & Jat, P. S. Establishment of conditionally immortalized epithelial cell lines from both colon and small intestine of adult H-2Kb-tsA58 transgenic mice. Proc Natl Acad Sci U S A 90, 587-91 (1993) and Xia, M. & Land, H. Tumor suppressor p53 restricts Ras stimulation of RhoA and cancer cell motility. Nat Struct Mol Biol 14, 215-23 (2007).
Treatment protocol
RNA was harvested from ten replicates for each cell population grown in non-permissive conditions for 48 hr, followed by 24 hr in media with 0% FBS to maximize the contribution of oncogenic signaling to gene expression. RNA was collected while cells were sub-confluent and all cell populations were actively cycling.
Growth protocol
Four polyclonal cell populations, control (Bleo/Neo), mp53 (p53175H/Neo), Ras (Bleo/RasV12) and mp53/Ras (p53175H/RasV12) were derived by retroviral infection of low-passage polyclonal young adult mouse colon (YAMC) cells. YAMC cells (a gift from R. Whitehead and A.W. Burgess) derived from the Immorto-mouse (aka H-2Kb/tsA58 transgenic mouse) expressing temperaturesensitive simian virus 40 large T (tsA58) under the control of an interferon γ-inducible promoter were maintained at the permissive temperature (33°C) for large T in the presence of interferon γ to support conditional immortalization in vitro. This permits expansion of the cells in tissue culture. In contrast, exposure of YAMC cells to the non-permissive temperature for large T (39°C) in the absence of interferon γ leads to growth arrest followed by cell death, indicating the absence of spontaneous immortalizing mutations in the cell population. The cells were cultured on Collagen IV-coated dishes (1μg/cm2 for 1.5 hr at room temp; Sigma) in RPMI 1640 medium (Invitrogen) containing 10% (v/v) fetal bovine serum (FBS) (Hyclone), 1×ITS-A (Invitrogen), 2.5 μg/ml gentamycin (Invitrogen), and 5 U/ml interferon γ (R&D Systems). All experiments testing the effects of RasV12 and p53175H were carried out at the non-permissive temperature for large T function (39°C) and in the absence of interferon γ.
Extracted molecule
other
Extraction protocol
Polysomal RNA was harvested from YAMC, bleo/neo, mp53/neo, bleo/Ras and mp53/Ras cells to obtain gene expression profiles reflective of protein synthesis rates. Cells were lysed in Extraction Buffer (50 mM MOPS, 15 mM MgCl, 150 mM NaCl, 0.5% Triton X-100 with 100 ug/mL cycloheximide, 1 mg/mL heparin, 200U RNAsin (2 uL/mL of buffer), 2mM PMSF). Supernatants were applied to 10-50% sucrose gradients, centrifuged at 36,000 rpm for 2 hr at 4°C and fractions were collected using an ISCO gradient fractionator reading absorbance at 254 nm. Polysome containing fractions were pooled and RNA was purified using the RNeasy Mini Kit (Qiagen) following the standard protocol for animal cells, except that sucrose fractions were mixed with 3.5 volumes Buffer RLT before binding to the RNeasy column. RNA was on-column DNase digested as part of the RNeasy RNA extraction protocol.
Label
biotin
Label protocol
Five micrograms of RNA was reverse transcribed and labeled using the mAMP kit (Ambion), with the 1x amplification protocol.
Hybridization protocol
The cRNA yield was fragmented and hybridization cocktails were prepared using Affymetrix standard protocol for eukaryotic target hybridization. Targets were hybridized to Affymetrix Mouse Genome 430 2.0 Expression Arrays at 45°C for 16 hours, washed and stained using Affymetrix Fluidics protocol EukGE-WS2v4_450 in the Fluidics Station 450.
Scan protocol
Arrays were scanned with the Affymetrix GeneChip Scanner 3000.
Description
Refer to submitted paper, McMurray et al.
Data processing
Expression values from the 50 microarrays processed were obtained using the RMA procedure with background correction in Bioconductor (http://www.bioconductor.org).
