Fig 6.

Signal integration by GR-KLF15 coherent feed-forward circuitry. (A) KLF15 mRNA expression following serum starvation in the presence or absence of the GR antagonist RU-486 (100 nM) in A549 and Beas-2B cells, as measured by qPCR. Bars represent fold induction (mean + SD) compared to that for cells treated with vehicle in complete medium. (B) qPCR analysis of KLF15, AASS, and PRODH mRNA expression in airway cells transferred to fresh complete or serum-free medium for 4 h prior to treatment with dex or vehicle for an additional 4 h. Transcript levels are expressed relative to that for cells receiving vehicle in complete medium. Bars indicate means + SDs. (C and D) ChIP-qPCR analysis of GR (C) and endogenous KLF15 (D) occupancy of AASS GBR1, PRODH GBR2, and MT2A GBR1 sites in Beas-2B cells that were serum starved for 4 h prior to treatment with dex for an additional 4 h. Factor occupancy is expressed relative to that of three negative-control sites, as described for Fig. 3. Data represent means + SDs expressed on a log₂ scale; values in parentheses indicate fold enrichment, on a nonlogarithmic basis, of occupied versus nonoccupied control sites, as described for Fig. 5. (E) Luciferase activity of the indicated reporters transiently transfected into Beas-2B cells prior to treatment with dex or vehicle in serum-free or complete medium. Bars indicate mean reporter activation (+ SD) relative to the activity of each construct in controls treated with vehicle in complete medium. *, P < 0.05. (F) Luciferase activity of the AASS GBR1 reporter cotransfected into Beas-2B cells with KLF15 or control siRNA prior to serum starvation in the presence or absence of dex. Reporter activity (mean + SD) is expressed relative to that in serum-starved cells treated with vehicle and siCtrl. *, P < 0.05.