Figure 7
SCL is required for mast-cell maturation. (A) Southern blot (left) and Northern blot (right) of mast-cell lines derived from SCL+/Δ or SCL−/Δ bone marrow cells. Tail DNA from MxSCL+/fl and MxSCL−/fl mice is shown to demonstrate the position of the SCLfl allele. The faint band running at 18S may represent cross-hybridization with LYL1. (B) Expression of the MC-CPA (CPA), MCP-5, and MCP-6 in SCL−/Δ mast-cell lines relative to SCL+/Δ mast-cell lines. Data represent analysis of at least 4 lines from each genotype. *P < .05 using Student t test. (C) Surface FcϵRI expression on resting (no IgE) or IgE-stimulated SCL+/Δ (shaded histogram) and SCL−/Δ (open histogram) mast-cell lines. The mean cell fluorescence (MCF) of FcϵRI expression was calculated from 4 cell lines of each genotype. The fold induction of FcϵRI expression by IgE compared with no IgE is shown for 4 lines of each genotype. (D) Ca2+ flux in SCL+/Δ or SCL−/Δ mast-cell lines determined by flow cytometry based on the change of FL5/FL4 ratio. Data are representative of 3 cell lines of each genotype. (E) Southern blot of a mast-cell line derived from an SCLfl/fl mouse. The cells were infected with a tamoxifen-inducible Cre recombinase retrovirus and treated for 48 hours with various concentrations of tamoxifen (0 to 3 μM). (F) Surface FcϵRI expression on Cre retrovirus-infected mast-cell lines derived from SCL+/fl mouse or SCLfl/fl mouse. FcϵRI expression was measured in the absence (open histogram) or presence (shaded histogram) of 1 μM tamoxifen. (G) Expression of the α-, β-, and γ- chains of FcϵRI in SCL−/Δ mast-cell lines relative to SCL+/Δ mast-cell lines. Data represent analysis of 8 cell lines of each genotype. (H) Spleen sections from SCL+/Δ and SCL−/Δ mice treated with 25 μg/kg per day mIL-3 (PeproTech) and 50 μg/kg per day rSCF (Amgen) for 5 days. Sections are stained with toluidine blue to detect cells with metachromatic mast-cell granules (→). Data represent the mean number of mast cells per high-power field from 2 mice. (I) The mean number of mast cells in peritoneal space from cytokine-treated mice shown in panel H was calculated by multiplying the absolute peritoneal cell count by the percentage of mast cells determined by flow cytometry: small c-kit+Sca-1− and large c-kit+Sca-1+.

SCL is required for mast-cell maturation. (A) Southern blot (left) and Northern blot (right) of mast-cell lines derived from SCL+/Δ or SCL−/Δ bone marrow cells. Tail DNA from MxSCL+/fl and MxSCL−/fl mice is shown to demonstrate the position of the SCLfl allele. The faint band running at 18S may represent cross-hybridization with LYL1. (B) Expression of the MC-CPA (CPA), MCP-5, and MCP-6 in SCL−/Δ mast-cell lines relative to SCL+/Δ mast-cell lines. Data represent analysis of at least 4 lines from each genotype. *P < .05 using Student t test. (C) Surface FcϵRI expression on resting (no IgE) or IgE-stimulated SCL+/Δ (shaded histogram) and SCL−/Δ (open histogram) mast-cell lines. The mean cell fluorescence (MCF) of FcϵRI expression was calculated from 4 cell lines of each genotype. The fold induction of FcϵRI expression by IgE compared with no IgE is shown for 4 lines of each genotype. (D) Ca2+ flux in SCL+/Δ or SCL−/Δ mast-cell lines determined by flow cytometry based on the change of FL5/FL4 ratio. Data are representative of 3 cell lines of each genotype. (E) Southern blot of a mast-cell line derived from an SCLfl/fl mouse. The cells were infected with a tamoxifen-inducible Cre recombinase retrovirus and treated for 48 hours with various concentrations of tamoxifen (0 to 3 μM). (F) Surface FcϵRI expression on Cre retrovirus-infected mast-cell lines derived from SCL+/fl mouse or SCLfl/fl mouse. FcϵRI expression was measured in the absence (open histogram) or presence (shaded histogram) of 1 μM tamoxifen. (G) Expression of the α-, β-, and γ- chains of FcϵRI in SCL−/Δ mast-cell lines relative to SCL+/Δ mast-cell lines. Data represent analysis of 8 cell lines of each genotype. (H) Spleen sections from SCL+/Δ and SCL−/Δ mice treated with 25 μg/kg per day mIL-3 (PeproTech) and 50 μg/kg per day rSCF (Amgen) for 5 days. Sections are stained with toluidine blue to detect cells with metachromatic mast-cell granules (→). Data represent the mean number of mast cells per high-power field from 2 mice. (I) The mean number of mast cells in peritoneal space from cytokine-treated mice shown in panel H was calculated by multiplying the absolute peritoneal cell count by the percentage of mast cells determined by flow cytometry: small c-kit+Sca-1− and large c-kit+Sca-1+.

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