ATF3 gene induction by homocysteine in HUVECs.

(A,B) Cultured HUVECs were incubated with 3 mmol/L homocysteine for the indicated time, and messenger RNA (mRNA) (panel A) and protein (panel B) level of ATF3 and GAPDH were measured by Northern blot and Western blot, respectively, as described in “Materials and methods.” The relative expression was shown in arbitrary units, and results were means of 3 independent experiments with SE bar. Bacterially expressed ATF3 protein was used as a control in Western blot (control). (C) ATF3 mRNA in the cells exposed to different concentrations of homocysteine for 4 hours was measured as in panel A. In lane 7, cells were preincubated with 10 μg/mL actinomycin D for 2 hours, then stimulated with 3 mmol/L homocysteine. (D) Nuclear run-on assay was performed with the use of nuclei from the homocysteine-stimulated cells for 1 and 2 hours, and their elongated transcripts were hybridized to ATF3 and GAPDH plasmid probes as described in “Materials and methods.”