Figure 4. CD99 engagement reduces the proliferation of RPMI8226 cells. (A) The number of RPMI8226 cells was counted using a hemocytometer after 3 days of treatment with an anti-CD99 Ab (YG32) or control IgG. The asterisk denotes a significant difference (p<0.05) between the two groups. (B) CFSE-labeled RPMI8226 cells at day 0 (filled histogram) were incubated for 3 days with control IgG (dotted line) or YG32 (solid line), and analyzed by flow cytometry. (C) RPMI8226 cells were cultured with YG32 or control IgG for 24 hours, stained with TMRE, and quantified using flow cytometry. (D) Annexin V-positive cells were quantified using flow cytometry after culture for the indicated amount of time in the presence of control IgG, YG32, or bortezomib. The ratio of the number of annexin V-positive cells to the total number of cells is shown (E) Expression of cyclin D1, D2, and D3 was measured by performing real-time PCR using the cDNA of RPMI8226 cells harvested after 3 days of incubation with YG32.