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. 2012 Jun 20;287(33):28067–28077. doi: 10.1074/jbc.M112.379487

FIGURE 3.

FIGURE 3.

MRTF-A is essential for BMP4-mediated inhibition of the TNFα/NF-κB p65 pathway. A, hPASMCs were transfected with 40 nm siRNA against MRTF-A or MRTF-B or non-targeting (control) siRNA control for 24 h, followed by treatment with or without TNFα (10 ng/ml) in the presence or absence of BMP4 (3 nm) for another 24 h. Total RNAs were extracted and subjected to qRT-PCR analysis. B, hPASMCs were infected with Adeno-p65 or GFP (control) cDNA, followed by transfected with 40 nm siRNA against MRTF-A or non-targeting siRNA control for 24 h, then treated with or without BMP4 (3 nm) for another 24 h. Total RNAs were extracted and subjected to qRT-PCR analysis. C, hPASMCs were infected with adenovirus carrying β-gal (control, Tet-Off), FLAG-tagged-mMRTF-A (Tet-Off) and Tet activator for 24 h. followed by treatment with or without TNFα (10 ng/ml) for another 24 h. Total RNAs were extracted and subjected to qRT-PCR analysis. D, hPASMCs were infected with adenovirus carrying β-gal, FLAG-mMRTF-A, and Tet activator, and co-infected with Adeno-p65 or GFP (control) cDNA as indicated for 48 h. followed by qRT-PCR analysis. E, medium was collected from hPASMCs culture infected with adenovirus carrying β-gal, FLAG-mMRTF-A and Tet activator, and co-infected with Adeno-p65 or GFP cDNA as indicated for 48 h, subjected to ELISA assay. F, PASMCs were infected with adenovirus carrying β-gal, FLAG-mMRTF-A and Tet activator, followed by transfection with NF-κB p65 or control plasmid. All samples were co-transfected with CXCL2 promoter construct (PSS2, −80/+76), subjected to luciferase assay. Luciferase activities were normalized to the β-galactosidase activities. G, hPASMCs were infected with adenovirus carrying β-gal, FLAG-mMRTF-A and Tet activator, followed by treatment of TNFα (10 ng/ml). Recruitment of NF-κB p65 to the IL-1β or CXCL2 promoter was examined by ChIP assay. H, PAC-1 cells were transiently transfected with or without expression constructs encoding Myc-tagged MRTF-A and FLAG-NF-κB p65, then stimulated with or without BMP4 (3 nm) in the presence or absence of TNFα (10 ng/ml) for 4 h. Total cell lysates prepared from these cells were subjected to immunoprecipitation with anti-FLAG monoclonal antibody. Immunoprecipitates were separated by SDS-PAGE, followed by immunoblot with anti-Myc antibody. Total cell lysates were immunoblotted with anti-Flag or anti-Myc antibody to visualize the expression levels of NF-κB p65 and MRTF-A.