Growth defect in AKO and AhR^d mast cells in response to IL-3. (A) The percentages of c-Kit⁺FcεRI⁺CD49b^- mast cells. Bone marrow cells were cultured in 30% WEHI-3-conditioned medium, and at indicated time points, aliquots of cultured cells from WT, AhR^d, and AKO mice were stained for c-Kit, FcεRI, and CD49b and analyzed by flow cytometry. *P < .05 (WT vs AKO). Data are representative of 3 independent experiments. (B) The relative level of c-kit analyzed by flow cytometry and measured as mean fluorescence intensity (MFI). Inset: c-kit expression detected by real-time reverse-transcription polymerase chain reaction from D31 mast cells.*P < .05. Data are representative of 3 independent experiments. (C) To determine the cell numbers, media were changed weekly and the numbers of suspension cells excluding trypan blue were recorded over 5 weeks. *P < .05 (WT vs AhR^d); ^#P < .05 (WT vs AKO). Data are representative of 3 independent experiments. (D) Mast cells were negatively selected on day 21 (D21) and day 30 (D30) after the initiation of the culture and stimulated with 5 ng/mL IL-3 for 3 days and the cell proliferation was evaluated with Cell counting kit-8. *P < .05. Data are representative of 3 independent experiments. BMMCs were first synchronized at G0/G1 in media devoid of cytokines for 16 hours and then stimulated with IL-3 (5 ng/mL) and/or SCF (20 ng/mL). The percentages of the cell population at (E) the S+G2/M and (F) G1 phases were determined at the 24-hour time point. *P < .05. Data are representative of 3 independent experiments. (G) Analysis of IL-3–induced signaling. Mature BMMCs from WT or AKO mice were first cultured in the cytokine-free media for 6 hours and then stimulated with 5 ng/mL IL-3 for different time points, followed by western blotting analysis of activation of STATs, AKT, and MAPKs in cell lysates by the use of the respective anti-phospho or total protein antibodies. Data are representative of 3 independent experiments.