Cell-cycle–dependent oscillation of endogenous GATA2 expression in P815 mast-cell line. (A-C) After culture in medium containing thymidine for 16 hours, P815 cells were washed twice and resuspended at 2 × 105 /mL in fresh medium containing nocodazole. Following a final 1-hour incubation with (C) or without (A,B) MG132, cells were harvested at each time point and divided into 3 portions for DNA content analysis, for immunoblot analysis, and for RNA extraction. Representative results of 3 independent experiments were shown. (B) RNA blot analysis of the thymidine-nocodazole experiment. (C) Immunoblot analysis after incubation with MG132 for last 1 hour. (D) Cells in confluent culture at 40 hours after passage were diluted with fresh medium to 2 × 105/mL and cultured for an additional 10 hours. (A; D, left panel) DNA content histogram. (A; D, right panel) Immunoblot analysis. (E) Chromatin immunoprecipitation experiments of P815 cells released from thymidine treatment into fresh medium lacking nocodazole. A representative result of 3 independent experiments is shown. (Left panel) Density plot analysis of GATA2 expression and DNA content. Horizontal and vertical lines represent borders of control IgG fluorescence and borders of 2N DNA, respectively. Numbers indicate the percentage of cells in each quadrant. (Right upper panel) Results of quantitative PCR analysis of GATA2 binding to Bcl-X promoter GATA sequences. Mean ± standard deviation of triplicate PCR experiments. Ig indicates results of immunoprecipitation using pre–immune rabbit IgG; (−), results of the culture without thymidine treatment; and 1/10Pr, DNA extracted from 1/10 input cell lysates. In negative control experiments, we did not detect significant levels of myogenin gene in immunoprecipitates with GATA2 antibody (data not shown). (Right lower panel) GATA2 expression demonstrated as Y geometric mean channel in density plot analysis.
