Rapmycin disrupts G-CSF–induced myeloid differentiation of mEB8-ER/mBB8-ER cells. (A-B) Top panel: Western blot of threonine-phosphorylated S6K (at residue 389: p-S6K) in mEB8-ER (A) or mBB8-ER (B) cells with or without rapamycin treatment. Cells were treated or not treated with G-CSF (2 ng, 30 minutes). Total S6K was a loading control. A typical experiment of 4 independent experiments. Bottom panel: Relative protein levels of p-S6K in mEB8-ER or mBB8-ER cells with various treatments. Quantification of blots from 4 separate experiments. Each bar represents the mean ± SEM. *P < .05. (C-D) Wright-Giemsa staining (top) and relative percentage of CD11b, Gr-1, CD16, and CD80-positive cells (bottom) in mEB8-ER (C) or mBB8-ER cells (D) measured with flow cytometry. Cells were induced to differentiate in the presence of G-CSF (2 ng/mL, 6 days) with (“G + Rap”) or without (“G-CSF”) rapamycin treatment (100nM). Four separate experiments were conducted, and quantification of 3 replicates of a typical experiment. Each bar represents the mean ± SEM (error bars). All values were normalized to the level (= 1) in cells without rapamycin treatment. *P < .05. Bar represents 10 μm.