KOBA cells repress <i>Cdkn</i> expression and enhance proliferation of OP9 cells by Notch activation.

<p>(<b>a</b>) Expression of indicated Notch target genes was determined in OP9, OP9/L, OP9/P, and KOBA cells were determined using quantitative RT-PCR. The means and SEs of triplicate determination. *<i>P</i> < 0.01. (<b>b)</b> Cell surface expression of Notch receptors and ligands was analyzed in OP9 and KOBA cells, respectively, using FACS. Shaded areas indicate control staining (left). C2C12 myoblast cells were co-cultured with or without KOBA cells for 8 days, and <i>Notch1</i> transcripts in isolated C2C12 were determined by quantitative RT-PCR (right). The means and SEs of triplicate culture. *<i>P</i> < 0.05. (<b>c</b>) OP9, OP9/L, OP9/P, and OP9 cells cultured with coated Dll4-Ig protein were lysed and immunoblotted with anti-Hes1 and anti-actin antibodies (upper). Arrowhead indicate Hes1 signal at 31 kDa, and relative signal densities of Hes1 over actin are shown. OP9 cells were co-cultured with KOBA cells in the absence or continuous presence of varying concentrations of DAPT for 8 days, and the isolated OP9 cells were lysed and immunoblotted with anti-Hes1 antibody (lower). Arrowhead indicate Hes1 signal, and relative signal densities of Hes1 over Actin are shown. (<b>d</b>) Expression of indicated <i>Cdkn</i> genes was determined in OP9, OP9/L, and OP9/L induced in the presence of 15 μM DAPT. The means and SEs of triplicate determination. (<b>e</b>) OP9 cells (1 × 10⁴ cells) were cultured alone or with KOBA cells in the absence or presence of 15 μM DAPT for 8 days and the viable cell numbers were counted (left). OP9 cells (1 × 10⁴ cells) were also cultured with or without coated Dll4-Ig for 8 days, and the viable cell numbers were counted (right). The means and SEs of triplicate culture are shown.