CNTO 530 activates known EPO signal transduction pathways in primary bone marrow proerythroblasts and induces sustained p70S6K activation. (A) CNTO 530 and EPO dose-dependent support of CFUe formation: To initially examine ex vivo effects on CFUe progenitors, adult murine bone marrow cells were plated in methyl cellulose colony-forming assays in the presence of various concentrations of CNTO 530 (0.08, 0.25, 1.0 μg/mL) or EPO (epoetin-alfa; 0.08, 0.25, 1.0 U/mL) for direct comparison. At 50 hours of culture, frequencies of 8- to 16-cell CFUe were determined; values are mean ± SE and are means of 3 35-mm culture dishes for each dose. (B-E) CNTO 530 and EPO modulation of p70S6K, AKT, STAT5, and ERK1,2 in primary bone marrow proerythroblasts: Primary erythroid progenitor cells from adult bone marrow were expanded in SP34EX medium. At 72 hours, Kit^posCD71^highTer119^neg proerythroblasts were then isolated. Cytokines were withdrawn and cells (at 10⁶ cells/mL) were incubated for 5.5 hours in IMDM, 0.5% BSA, 15 ng/mL insulin, and 0.1 mM 2-ME. Cells next were exposed in parallel to either CNTO 530 (0.5 μg/mL) or EPO (2 U/mL) for 0, 10, 20, 40, and 80 minutes. Levels of activated phospho-T421/S424-p70S6K (A); phospho-S473-AKT (B); phospho-Y694-Stat5 (C); and phospho-T202/Y204-ERK1,2 (D) then were determined. Quantitation was by Image-J analysis and was normalized based on levels of total p70S6K, AKT, STAT5, and ERK1,2. Values are percentage maximal signals for CNTO 530 and EPO.