Fig. 5.

Hydrogen peroxide up-regulates Msx2 promoter activity. A, upper panel, TNF treatment stimulates the Msx2 promoter-luciferase reporter activity in transiently transfected C3H10T1/2 mesenchymal cells via proximal promoter elements. A, lower panel, peroxide treatment also stimulates Msx2 promoter-luciferase reporter activity via proximal promoter elements. By contrast, the RSV (Rous sarcoma virus) proximal promoter was not stimulated by peroxide. Treatments were for 6 h, n = 3 per treatment group, and replicated more than three times. B, Upper-strand sequences of the Msx2 proximal promoter duplex oligos radiolabeled and used as EMSA probes. C, H₂O₂ treatment up-regulates DNA binding activity recognizing the proximal Msx2 promoter regions −69/−40 and −11/+26 detected by gel shift assay. Brackets indicate the EMSA complexes visualized by autoradiography. The unbound/unshifted radiolabeled duplex probes have been run off the bottom of the gel. Treatments were for 4 h. D, Upper-strand sequences of the native (WT) and mutant (M1–M5) Msx2 proximal promoter duplex oligos used for cold competition studies. E, Cold competition studies assessing binding specificity of complexes recognizing the Msx2 promoter region −69 to −40 (lanes 1–5) and −11 to +36 (lanes 6–10). The unbound, unshifted radiolabeled duplex probes are seen at the bottom of this gel (arrow). **, P < 0.01 vs. vehicle.