FIG. 7.
In vivo inactivation of FoxO1a transcriptional activity by a constitutively active form of cGKIα. (A) Luciferase activity in proliferating or differentiating C2C12 myoblasts is shown after transient transfection with a luciferase reporter containing the +6.5/+6.8-kb cGKIα enhancer element alone or in the presence of cGKI-CA, wild-type FoxO1a (FoxO1a-WT) plus cGKI-CA, or FoxO1a-cGKIMut.A plus cGKI-CA. cGKI-CA does not inhibit the transcriptional up-regulation of the reporter by FoxO1a-cGKIMut.A, while it completely inhibits the up-regulation by endogenous or overexpressed FoxO1a-WT. The +6.5/+6.8-kb cGKIα enhancer element without (w/o) the FoxO binding sites is shown as a negative control. (B) Immunofluorescence of cGKI-CA-transduced C2C12 cells shows a reduction in overall cell size and dramatic changes in morphology as well as cytoplasmic accumulation of FoxO1a. Transduced cells are indicated by arrowheads, while wild-type cells are not. An anti-GFP antibody demarcates transduced cells. 4′,6′-Diamidino-2-phenylindole (DAPI) staining is used to show the cell nucleus.