BMP4 enhances ITGA4 expression via p38 phosphorylation in KLS/CD150⁺CD48⁻ cells within 6 hours. (A-B) Lin⁻ BM cells were cultured in serum free medium in the presence of SCF+TPO in the presence or absence of BMP4 alone or with SB203580 for 5 days. ST = SCF/TPO; STB = SCF/TPO/BMP4; STBS = SCF/TPO/BMP4/SB203580. (A) After 5 days, flow cytometry was performed to analyze p38 MAPK phosphorylation as well as ITGA4 expression. The cells were harvested and stained with antibodies against Lineage positive cells, CD48, CD150, Sca-1, and c-Kit combined with anti–phospho-p38 MAPK or ITGA4 antibodies. CD48⁻CD150⁺KLS cells were gated (top panel) and the levels of phosphorylated p38 MAPK (bottom left) and ITGA4 expression (bottom right) were compared among different conditions. Representative example of 5 experiments. (B)The CD48⁻CD150⁺KLS subpopulation was sorted from Lin⁻ BM cell progeny cultured under the different conditions. Immunostaining was performed to evaluate p38 MAPK and MITF activation using antibodies against phospho-p38 MAPK and MITF. Representative example of 3 experiments. (C) Fresh KLS cells were cultured in the presence of SCF+TPO with or without BMP4 for 3 hours (top panel) and 6 hours (bottom panel). Flow cytometry was performed to analyze phosphorylation status of MAPK p38 (left) and ITGA4 (right) expression. Representative example of 4 independent experiments. (D) Homing potential of KLS cells cultured for 6 hours in SCF+TPO with or without BMP4 was compared with freshly isolated KLS cells (n = 8; *P = .002).