P1A as an Oncogene Figure 4. Enhanced proliferation and progenitor activity of P1A Tg bone marrow. A. BrdU incorporation by flow cytometry; representative profiles of BrdU incorporation in bone marrow cells. B. The proportion of BrdU positive cells among bone marrow cells, showing mean 6 SD of BrdU positive cells (n = 3, two-tailed unpaired Student’s t-test). C. Colony-forming cell (CFC) assay. There was a higher colony number in P1A+Rag22 compared to Rag22 bone marrow cells. The cells cultured in second round were taken from the first round culture (n = 3, two-tailed unpaired Student’s t-test). D. Higher colony number in all colony types, including burst-forming unit-erythroid (BFU-E), colony-forming unit granulocyte (CFU- G), colony-forming unit macrophage (CFU-M) and colony-forming unit granulocyte macrophage (CFU-GM), were seen in P1A+Rag22 than in Rag22 by inverted microscopy. (n = 3, two-tailed unpaired Student’s t-test) doi:10.1371/journal.pone.0013439.g004 containing 15% FBS, 0.1 mM b-mercaptoethanol, 103 u/ml leukemia inhibitory factor, and 4 mM glutamine. Rag22/2 mice were obtained from the Taconic Laboratories (Terrytown, NY). P1A transgenic (P1A Tg) mice were as described previously [6,9]. The transgene has been crossed to BALB/c for more than 10 generations, and was crossed to BALB/c Rag22/2 background for the current studies. P1A+Rag22/2 mice in this study came from 7 breeding pairs. All animal experiments were conducted in accordance with accepted standards of animal care and approved by the Institutional Animal Care and Use Committee of University of Michigan. Western blot analysis Samples of mouse tissues or cells were lysed in protein lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% NP-40) and protease inhibitor cocktails (Sigma) including 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, aprotinin, bestatin, E-64, leupeptin and pepstain A were added. Cell lysates were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electropho- resis, transferred to polyvinylidene fluoride (PVDF) membranes, and incubated with anti-P1A rabbit antibody specific for the peptide sequence, YEMGNPDGFSP (Genemed Synthesis, 1:500 PLoS ONE | www.plosone.org 5 October 2010 | Volume 5 | Issue 10 | e13439