ERRα represses osteoblast differentiation markers.

<p><b>A.</b> Evaluation of the expression of osteoblast differentiation markers by real time PCR at the indicated time after differentiation of mouse calvarial cells. ALP: alkaline phosphatase; Ocn: osteocalcin; Opn: osteopontin. A single representative experiment (out of three, performed in triplicate) is shown. Data are expressed relative to differentiation day 1 with error bars representing S.D. <b>B.</b> Reintroduction of ERRα in cKO cells normalizes the expression of differentiation parameters. Lentivirus encoding human ERRα was used to infect mouse calvarial cells induced in osteoblastic differentiation. RNAs were extracted after differentiation day 5. Data are expressed relative to control-infected cells (empty lentivirus: c) as mean +/− s.e.m. Differences for each gene are significant (<i>p</i><0.005) except where indicated. Experiments were performed in triplicate with n = 4. <b>C.</b> ERRα represses Runx2 transcriptional activities. C3H10T1/2 cells were transfected with OSE6-Luc (Runx2-responsive) or ERRE-Luc (ERRα-responsive) plasmid together with the indicated amount of Runx2, and fixed amounts of ERRα or ERRγ, as indicated. CMV-renillaLuc was added as a transfection efficiency control and PGC-1α-encoding plasmid was added together with ERR for the right panel as indicated. Results are expressed as fold activation over transfection with activator and expressed as the average of a typical triplicate experiment (out of three) with error bars representing S.D. *: <i>p</i><0.05; **: <i>p</i><0.01; ***: <i>p</i><0.005; ns: non significant.