D609 inhibits transcriptional activation of the type II NOS gene in human and mouse cells
(A) Mouse RAW 264.7 cells were activated with 1 µg/ml LPS for 5 hr, and RNA was collected. An aliquot of 50 µg of total RNA was hybridized with a mouse type II NOS probe prepared from a 418-base, radiolabeled fragment (position 54–471 from the sequence of Xie et al. ; Genbank accession number M87039), or a loading control consisting of a radiolabeled, 315-base fragment specific for mouse GAPDH. Following RNase digestion, the mixture was resolved on a Polyacrylamide gel and subjected to autoradiography; the results are presented in Fig. 3A. Lanes 1 represent the protected fragments derived from unactivated cells, lanes 2 were derived from LPS-activated cells, and lanes 3 were derived from activated, D609-treated cells. (B) Human A549 cells were activated; total RNA was isolated and hybridized to a human type II NOS radiolabeled probe derived from a 179-base sequence of human type II NOS (position 2946-3124 reported by DA Geller et al. ; Genbank accession number L09210). All other experimental protocols were as described for the mouse RNase protection assay. The data are presented in B. (C) The type II NOS-specific bands (indicated by arrows) were excised from the gel, and radioactivity was quantified with a gamma counter. The data were normalized to percent of type II NOS radioactivity divided by the radioactivity present in the GAPDH loading control lane. Figure 3C presents the results of the quantitation of protected RNA isolated from control and treated murine cells. (D) Human type II NOS-protected fragments were quantified as described for murine sequences in Fig. 3C. The loading control was a 249-radiolabeled base sequence specific for human β-actin. Data are presented as described for Fig. 3C.