Growth factor signaling cannot rescue neutropenia in Mcl-1–deleted mice. (A) Representative flow cytometric analysis of blood from littermate control (Mcl-1^f/wt plus LysM-Cre) and Mcl-1–deleted mice (Mcl-1^f/null plus LysM-Cre) treated with G-CSF (250 μg/kg) or vehicle control for 5 days. Top right quadrant represents Mac-1 and Gr1 double-positive neutrophils. (B) Percentage of apoptotic mature and immature neutrophils (Mac-1⁺, Gr1^{(high and Intermediate)}, annexin-V⁺) from littermate control (Mcl-1^f/wt plus LysM-Cre) and Mcl-1–deleted (Mcl-1^f/null plus LysM-Cre) BM cells cultured in the presence or absence of GM-CSF (10 ng/mL). Data presented are the average of 3 independent experiments, and the error bars represent the SEM. (C) Flow cytometric analysis of BM from littermate control (Mcl-1^f/wt plus LysM-Cre) and Mcl-1–deleted mice (Mcl-1^f/null plus LysM-Cre) cultured in GM-CSF (10 ng/mL) for 1 day. Samples were stained with Mac-1 and Gr1. Top right quadrant designated as mature neutrophils; bottom right quadrant, immature neutrophils. (D) MCL-1 protein levels as determined by immunoblot analysis of BM cells from control mice (Mcl-1^f/wt and Mcl-1^f/wt plus LysM-Cre) or Mcl-1–deleted mice (Mcl-1^f/null plus LysM-Cre) cultured with or without GM-CSF (10 ng/mL) at indicated time points. Blots were probed for PDI expression to serve as a loading control. (E) In vitro myeloid differentiation of Mcl-1^f/wt littermate control (■) or Mcl-1^f/null plus LysM-Cre–deleted mice (red bar) cultured in G-CSF, M-CSF, or GM-CSF (10 ng/mL). After 48 hours, cells were harvested, cytospun, and subjected to manual BM differential for myeloid precursors and neutrophils. Graphs represent the average of 3 individual experiments; error bars denote SEM.