Figure 5.

DEX inhibits Bmp2 expression via far-downstream regulatory element(s). MC3T3-E1 cultures were stably transfected with Bmp2-LacZ BAC reporter constructs, and treated with 1 μM DEX starting on day 3. (A) Schematic illustration of the 3′ and 5′ BAC constructs. (B) X-gal staining of days 4–11 cultures stably transfected with the 3′ BAC. Similar staining was seen in three independent 3′ BAC lines. Two independent lines that were stably transfected with the 5′ BAC construct did not stain for β-gal at any time point; only day 11 is shown for one of the 5′ BAC lines. (C) Micrographs of cultures from B, showing X-gal staining of condensing areas in CONTROL cultures, and sparse staining in DEX-treated cultures that failed to condense. Original magnification 100 ×. (D) Biochemical analysis of β-gal activity in day 11-cultures carrying 3′ and 5′ Bmp2-BAC constructs. β-gal activity was corrected for total protein. β-gal activity of non-transfected MC3T3-E1 cultures was negligible. Values represent the mean ± SD of triplicate cultures. *p < 0.05 compared to CONTROL.