Transcription activation of 9E3/cCAF by thrombin.

(A) Northern blot analysis of cCAF mRNA in primary fibroblasts after thrombin stimulation. Total RNA was prepared using TRIzol reagent (GIBCO/BRL); RNA samples (20 μg each) were denatured in formamide-formaldehyde loading buffer containing ethidium bromide and separated on 1% formaldehyde-agarose gel. The blots were probed with a radiolabeled DNA probe prepared from the 9E3/cCAF complementary DNA (cDNA). The higher molecular weight RNA represents the full length of the gene (see accession no. AJ009800 or NID g4467411 in GeneBank). The bottom panel shows ethidium bromide staining of the 28S ribosomal RNA (rRNA). (B) Northern blot analysis of cCAF mRNA in primary fibroblasts after treatment with thrombin in the presence of the inhibitor of transcription actinomycin D (ActD) or the inhibitor of the actin cytoskeleton, cytochalasin D (Cyto D). The cells treated with Act D did not produce the higher molecular weight message, whereas those treated with Cyto D did. (C) Primary fibroblasts were cotransfected with 4 μg of the reporter plasmid containing the 1503-bp promoter region and 2 μg of PCH110 vector containing lac-Z as an internal transfection control. Thirty-six hours after transfection, the cells were treated in the presence or absence of thrombin for 3 hours and the extracts assayed for luciferase activity. Thrombin stimulation induced a significant increase in transcription. The background seen with the construct in absence of thrombin treatment is due to the stress generated by the transfection procedure. The data shown here are representative of several experiments performed under the same conditions but with different batches of primary fibroblasts. Bars represent SEMs of 3 samples per condition in the same batch of cells.