Fig. 5.

Two RFX5 mutants behaved as dominant negative factors activating collagen transcription. A, top, schematic structures of wild type as well as two mutated RFX5 constructs; bottom, wild type RFX5, RFX5Δ1, and RFX5Δ5 as well as LacZ constructs were expressed in IMR-90 cells. IMR-90 cells were infected with virus carrying RFX5, RFX5Δ1, RFX5Δ5, or LacZ viral expression construct, or an empty vector (TOPO). 48 h after infection, cells were harvested and whole cell extracts were prepared as described under “Materials and Methods.” Protein expressions were examined by 10% SDS gel followed by Western blot using an anti-V5 antibody. B and C, RFX5Δ1 and RFX5Δ5 both acted as dominant negative proteins activating basal level as well as IFN-γ mediated COL1A1 (B) and COL1A2 (C) expression. IMR-90 cells were infected with or without virus carrying RFX5, RFX5Δ1, RFX5Δ5, or LacZ viral expression construct, or an empty vector (TOPO). 48 h after infection, cells were treated with 100 units/ml IFN-γ ([unk]) or without (□) for an additional 24 h before harvesting. COL1A1 (B) or COL1A2 (C) mRNA was amplified using primers listed in Table III by real-time PCR. Each experiment was repeated three times in duplicate wells and data are expressed as relative RNA levels compared with no infection control. One-sample two-tailed t test (without IFN-γ) or one-way analysis of variance (with IFN-γ) was employed to assess the statistical significance (*, p < 0.05; **, p < 0.01). D, RFX5Δ1 and RFX5Δ5 both blocked the IFN-γ stimulated expression of HLA-DRA molecules. IMR-90 cells were infected with or without virus carrying RFX5, RFX5Δ1, RFX5Δ5, or LacZ viral expression construct, or an empty vector (TOPO). 48 h after infection, cells were treated with 100 units/ml IFN-γ ([unk]) for an additional 24 h before harvesting. HLA-DRA mRNA was amplified using primers listed in Table III by real-time PCR. Each experiment was repeated three times in duplicate wells and data are expressed as relative RNA levels compared with no infection control. One-sample two-tailed t test was employed to assess the statistical significance (***, p < 0.01). E, lentiviral infections did not lead to detectable changes in expression of CIITA mRNA. IMR-90 cells were infected with or without virus carrying RFX5, RFX5Δ1, RFX5Δ5, or LacZ viral expression construct, or an empty vector (TOPO). 48 h after infection, cells were treated with 100 units/ml IFN-γ ([unk]) or without (□) for an additional 24 h before harvesting. CIITA mRNA was treated an additional 24 h before harvesting. HLA-DRA mRNA was amplified using primers listed in Table III by real-time PCR. A representative experiment was shown.