Chk1/MEK1/2 inhibition induces caspase-3 activation and γH2A.X expression in statin-positive (G₀/G₁) MM cells. (A) H929 cells were incubated in 0.1% FBS medium for 64 hours, and then exposed to 1.5μM AZD6244 for 24 hours, followed by 300nM AZD7762 for an additional 18 hours. (B) Primary CD138⁺ MM cells isolated from the BM sample of a patient with MM were exposed to 5μM AZD6244 and 250nM AZD7762 for 24 hours. For panels A and B, cells were immunofluorescently stained with Abs against statin (red fluorescence) and cleaved (activated) caspase-3 (green fluorescence) and counterstained with DAPI (blue) after treatment. Images were captured microscopically at 60×/1.40 under oil and then merged as indicated. Arrows indicate cells exhibiting colocalization of statin and activated caspase-3 expression. Results are representative of 3 separate experiments. (C) H929 cells enriched for G₀/G₁ as described in panel A were exposed to MEK1/2 inhibitors (1.5μM AZD6244 or 1.5μM PD184352) for 24 hours, followed by Chk1 inhibitors (300nM AZD7762 or 300nM CEP3891 for an additional 12 hours. Flow cytometric analysis was used to quantify the percentage of cells coexpressing statin and activated caspase-3 (upper right quadrant). In parallel, untreated cells were incubated with IgG instead of statin or cleaved caspase-3 Abs as a negative control to demonstrate the specificity of the immunostaining. Numbers indicate the percentage of activated caspase-3–negative (blue) or –positive (red) in the statin⁺ population. Two additional studies yielded equivalent results. (D) Alternatively, G₀/G₁–enriched H929 cells were treated 1.5μM AZD6244 for 24 hours, followed by 300nM AZD7762 for an additional 6 hours, immunofluorescently stained with Abs against statin (red fluorescence) and γH2A.X (green fluorescence), and then counterstained with DAPI (blue). Images were captured microscopically at 60×/1.40 under oil and then merged as indicated. Arrows indicate cells coexhibiting statin and γH2A.X expression/foci formation.