FIG. 8.

Induction of integrated and endogenous target genes by OTT-MAL. NIH 3T3 cells harboring an integrated 3D.A-FosHA reporter gene whose expression requires proper splicing were immunostained with anti-HA antibodies (red) and Hoechst 33258 (blue). As controls, cells were either left untreated (A, top) or serum stimulated for 1 h (A, bottom). In panel B, cells were transiently transfected with either Flag-tagged OTT-MAL or OTT-MALΔRRM, kept under serum-starved conditions, and subsequently coimmunostained with anti-Flag antibodies (green), anti-HA antibodies (for detection of reporter gene expression; red), and Hoechst 33258 (blue). (C) Induction of smooth muscle α-actin by MAL, OTT-MAL, and OTT-MALΔRRM. Expression in stably transfected HEK293 clonal cells was induced by doxycycline (Dox; 1 μg/ml, 24 h), and the mRNA was isolated and subjected to quantitative RT-PCR with primers specific for acta2 and alas1. Shown is the average induction of acta2 compared to mock-transfected control cells after normalization to alas1 (error bars indicate the half range). The expression levels of the MAL, OTT-MAL, and OTT-MALΔRRM proteins in the various clonal lines are visualized in panel D by immunoblotting (IB) against HA. The values on the left are molecular sizes in kilodaltons.