Upregulation of mMCP-1 expression in mBMMC cultures after the addition of TGF-β1. Four flasks of mBMMC were cultured in the presence of WEHI/rrSCF/rmIL-9 (5 ng/mL) for 7 days to produce greater than 95% mast cells which were 20% ± 4% mMCP-1⁺. They were split into 16 separate flasks at 5 × 10⁵ mBMMC/mL, and supplemented with WEHI/rrSCF/rmIL-9 to which was added either vehicle alone or rhTGF-β₁ (1 ng/mL of culture supernatant). (A) shows the percentage of mMCP-1⁺ mast cells from mBMMC cultures with (□) or without (▪) addition of TGF-β1 on day 7. Data are from quadruplicate cultures except where stated. (B) shows the concentrations of mMCP-1 in culture supernatants (in nanograms per milliliter). (C) shows the concentration of mMCP-1 in cell pellets (in nanograms per 10⁶ cells) from these cultures 7 days after the addition of rhTGF-β₁ (▪) or in rmIL-9/WEHI/rrSCF (□). (D) is a Western blot to show mMCP-1 expression in mBMMC grown in rhTGF-β₁/rmIL-9/WEHI/rrSCF (TGF-β1) compared with an equivalent loading from mBMMC grown in rmIL-9/WEHI/rrSCF (IL-9) as detected by MoAb RF 6.1. A lane containing purified mMCP-1 (M1) was included as a control. Extracts from 2.5 × 10⁴ mBMMC were loaded in the other lanes. Molecular weights in kilodaltons are shown. (E) shows the RT-PCR products of chymase genes from total RNA extracted from mBMMC cultures. RNA was from triplicate mBMMC cultures (A, B, and C) in rhTGF-β₁/rmIL-9/WEHI/rrSCF (T/I/W/S) or rmIL-9/WEHI/rrSCF (I/W/S) (7 days after addition of TGF-β₁). Initial dilutions of the RNA template before reverse transcription are indicated (1, 0.1, 0.01, and 0.001 μg/mL). Primer sets used for PCR were specific for the mMCP-1, mMCP-2, mMCP-4, mMCP-5, and β-actin genes as indicated.