PD0332991 affects entry into the cell cycle and proliferation of AML model cell lines. (A) Cell-cycle analysis. Flow cytometric analysis of MOLM13 and U937 cells treated with DMSO (vehicle control) for 120 hours and PD0332991 (500 nM) for 24 and 120 hours and stained with propidium iodide. (B) Bar graph representation of cell-cycle analysis of MOLM13, MV4-11, THP-1, and U937 cells treated for 24 (□) and 120 hours (▒) with PD0332991 (500 nM). The cells were stained with propidium iodide and analyzed by flow cytometry. The percentage of cells in S/G₂ compared with the control cells, which were normalized to 100% (treated with DMSO for 24 and 120 hours, respectively), was plotted (mean ± SE of 3 independent experiments). (C) Proliferation assay. MV4-11, MOLM13, THP-1, and U937 cells were plated in the presence of DMSO (vehicle control) or PD0332991 (500 nM) at a density of 0.5 × 10⁶ / mL. The cells were counted every 2 days, and the cell number was adjusted to 0.5 × 10⁶/ mL (mean ± SE of 3 independent experiments). (D) MTT assay. MV4-11, MOLM13, U937, THP-1 cells, as well as primary patient blasts from 2 patients (Pt 17 [Flt3 ITD] and Pt 75 [Flt3 wt]) were incubated in triplicate for 3 days with DMSO (control), PD0332991 (500 nM) (░), SU14813 (30 nM for cell lines and 100 nM for patient samples) (▒), or PD0332991 (500 nM) plus SU14813 (30 nM for cell lines and 100 nM for patient samples) (▓). The DMSO control is normalized to 100% (mean ± SE of 3 independent experiments for the cell lines and one experiment with triplicate data points for the patient samples). (E) Apoptosis assay. MV4-11, MOLM13, THP-1, and U937 cells were incubated for 3 days with DMSO (control), PD0332911 (250 nM) (░), SU14813 (100 nM) (▒), or PD0332991 (250 nM) plus SU14813 (100 nM) (▓). The DMSO control is normalized to 100%. The cells were stained with Annexin V-PE and 7-AAD followed by flow cytometric analysis (see Figure S4 for primary data).