Chk1/MEK1/2 inhibitor regimens induce cell death in G₀ MM cells characterized by the Hst⁺/PY⁻ phenotype. (A) RPMI8226 cells were incubated in 0.2% or 10% FBS medium for 72 hours, after which time flow cytometry was performed to assess G₀ populations by double staining DNA with Hst and RNA with PY. The G₀ population (2N DNA/low levels of RNA, Hst⁺/PY⁻) was discriminated from the G₁ population (2N DNA/high levels of RNA, Hst⁺/PY⁺), whereas the S and G₂/M populations displayed > 2N DNA. G₀ cells (R17) were gated-in for further analysis. Compared with those cultured in 10% FBS (i), flow cytometric profiles indicate enrichment of G₀ population in cells cultured in 0.2% FBS (ii). In parallel, PI staining and flow cytometry were performed to monitor cell-cycle distribution (inset). Values indicate the percentage of cells in each phase. G₀/G₁–enriched 8226 cells were then exposed to MEK1/2 inhibitors (1.5μM AZD6244 or 1.5μM PD184352) for 24 hours, followed by Chk1 inhibitors (400nM AZD7762 or 500nM CEP3891) for an additional 12 hours. Flow cytometric analysis was performed to assess cell death (7-AAD positivity, R16) in the G₀ (Hst⁺/PY⁻, R17 gate-in) population (iii-v). To ensure specificity of this assay, control experiments were performed in parallel, including negative controls (without fluorescent dye), individual staining with each dye, and double staining with each pair of dyes. Results are representative of 3 separate sets of experiments. (B) H929 cells cultured in 10% FBS medium reached a density of 1 × 10⁶ cells/mL, after which time viable SSP cells were sorted by exclusion of Hst staining of MM cells, as described in supplemental Methods. In brief, cells were incubated with Hst and then stained with PI. For flow cytometric analysis, after gating out PI⁺ dead cells (i), gates were set for both the SP (ii) and low FSC (small size) population (iii). In parallel, cells were coincubated with 50μM verapamil (iv), which blocks Hst efflux, as a control. The cell-cycle profile was determined by PI staining immediately after sorting (v top, unsorted cells; v bottom, sorted SSP cells). (C) Sorted SSP cells in 5% FBS medium were then treated with the AZD6244/AZD7762 regimen for 14 hours and stained with activated caspase-3 plus PI to determine the percentage of apoptotic cells within the G₀/G₁ population. Two additional experiments yield roughly identical results.