In vivo occupancy of MS7 and influence of RUNX3 on CD11a cell surface expression. (A) Nuclear extracts were obtained from U937 and 2 independent clones (nos. 6 and 8) of U937-Runx3 cells; 10 μg from each extract was subjected to Western blot using polyclonal antisera-specific Runx3.47 The position of the Runx3 protein is indicated. (B) CD11a/CD18 and CD11c/CD18 cell surface expression on untransfected U937 and 2 independent U937-Runx3 cell clones, as determined by flow cytometry using the monoclonal antibodies TS1/11 (anti-CD11a) and HC1/1 (anti-CD11c). Negative control fluorescence was determined using the supernatant from the T3b hybridoma (anti-CD3). Data represent mean ± SD of the mean fluorescence intensity values obtained in 4 independent experiments (*P < .002 and P < .000 05 for CD11a expression in clone nos. 6 and 8, respectively, compared with the CD11a expression in untransfected U937 cells). (C) Chromatin immunoprecipitations on uninduced (THP-1) or PMA-differentiated (96 hours) THP-1 cells (THP-1+PMA), using antibodies specific for C/EBPα, RUNX3, CD40 (negative control), or no antibody. Precipitated chromatin was analyzed by PCR using a pair of CD11a promoter-specific primers that flank the MS7 element and amplify a 221 bp DNA fragment. Input lane represents the PCR analysis performed on the DNA precipitated from a 1:20 dilution of the starting sonicated lysate. Each experiment was performed twice with similar results, and 1 of the experiments is shown.
