Ronai et al. 10.1073/pnas.0706057105. |
Fig. 5. Lysates from Fig. 3f were subjected to Western blotting for cyclin A and cyclin B1. Two mice for each group are shown. b-Actin was used as loading control.
SI Methods
Preparation of Epidermal Cell Lysates.
The dorsal skin of the mice was excised and placed on a glass plate on ice, and the epidermis was removed with a razor blade and placed into RIPA lysis buffer. The lysis buffer contained 25 mM Tris·HCl (pH 7.6), 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, 1 mg/ml aprotinin, 1 mM Na3VO4, and 1 mM NaF. The lysates were incubated on ice for 10 min, snap frozen in liquid nitrogen, rethawed, and then centrifuged at 14,000 ´ g for 15 min at 4°C.BrdU Incorporation.
Eight-week-old K14.ATF2f/f mice and wild-type (WT) mice were shaved 2 days before treatment with phorbol 12-tetradecanoate 13-acetate (TPA) (10 mg in 200 ml of acetone). BrdU (0.1 mg/g of body weight) was injected i.p. 1 h before the mice were killed. The mice were killed 7, 24, and 48 h after six TPA treatments; their dorsal skins were removed, fixed in formalin, and processed for paraffin embedding. Immunohistochemical staining was performed with a monoclonal anti-BrdU antibody (1:100; Sigma-Aldrich), which was applied to each section overnight at 4°C after masking mouse IgG according to the protocol of the M.O.M. kit. Biotinylated anti-mouse IgG was used as the secondary antibody, and the immunoreaction was visualized by the avidin-biotin-peroxidase complex immunostaining method with diaminobenzidine as the substrate. BrdU-positive and -negative basal cells were counted in three to five randomly selected areas of each skin section (three to four sections per mouse), and the mean percentage of BrdU-positive cells and standard deviation (SD) for each treatment group were determined.FACS Analysis
. Primary keratinocytes of WT and K14.ATF2f/f mice were cultured for 3 days before cells were trypsinized, washed, and fixed (70% ethanol in PBS). The cells were stained with propidium iodide (Sigma) and analyzed by using a FACSCanto cell sorter with CELLQUEST software (Becton Dickinson). To determine percentages of cells in the G1, S, and G2/M phases, original data were analyzed by using ModFit LT software.Microarray and Real-Time RT-PCR Analyses.
RNA from papillomas was isolated with TRIzol reagent (Sigma). Pooled RNA from the papillomas of two WT and three K14.ATF2f/f papillomas were then hybridized in triplicate to the Sentrix Mouse-6 Expression BeadChip (Illumina) according to the manufacturer's recommendations. Data were quantile-normalized by using the BeadExplorer bioconductor package. A t test was performed by using the R (a language and environment for statistical computing statistics package) Development Core Team (www.R-project.org) to determine evidence for differential expression. P values were corrected for multiple testing (1). To confirm differences in gene expression, real-time RT-PCRs were performed by using Stratagene Mx3000p.RNAs from WT and K14.ATF2f/f papillomas were isolated with TRIzol reagent. These RNAs were converted into Cy3- and Cy5-labeled cDNAs and hybridized with the Sentrix Mouse-6 Expression BeadChip, according to the manufacturer's recommendations. To confirm differences in gene expression, real-time RT-PCRs were performed by using Stratagene Mx3000p.
Assessment of Staining in Skin Tumors TMA.
A skin cancer tissue array was purchased from US Biomax, Inc. The percentage of ATF2-positive tumor cells was determined semiquantitatively by scoring the intensity of the immunostaining (2). The intensity was graded as: 0, negative; 1+, weak; 2+, moderate; 3+, strong. The immunoscore was determined by visual inspection (intensity ´ % of tumor stained = 300 as a maximal score translated as 3 ´ intensity in 100% of the tissue). Scores were then divided into four categories: 1, scores ranging from 0 to 75; 2, scores ranging from 76 to 150; 3, scores ranging from 151 to 225; and 4, scores ranging from 226 to 300. Analysis was performed by three readers at Burnham Institute and Yale University. The slides were scanned with the slide scanner at ´20 resolution (Aperio's ScanScope CS), and the images were retrieved with Imagescope viewing software.Soft Agar Assay.
Keratinocytes derived from WT and K14.ATF2f/f newborn mice were infected with an H-RasV12 retrovirus. Sixteen hours after infection, 5 ´ 103 cells were trypsinized to form a single-cell suspension and seeded in triplicate onto six-well plates in MEM containing 0.35% agarose overlying a solidified 0.7% agarose. After the cell-containing layer solidified, 0.7% agarose was overlayed. Fresh growth medium was added every 4 days. Plates were incubated at 37°C in 5% CO2 for 21 days. Colonies ([mt]0.5 mm) were counted under the microscope.1. Benjamini Y, Yekutieli D (2001) Ann Statist 29:1165-1188.
2. Krajewska M, Kim H, Kim C, Kang H, Welsh K, Matsuzawa S, Tsukamoto M, Thomas RG, Assa-Munt N, Piao Z, et al. (2005) Clin Cancer Res 11:5451-5461.