FIGURE 1. Pax7-FKHR represses MyoD transcriptional activity.

A. MyoD ability to direct C3H10T1/2 cells into the myogenic lineage (left panel) is efficiently inhibited by Pax7-FKHR (middle panel) co-expression, analogous to the previously described effect for Pax7 (right panel). Myogenic conversion was evaluated trough the co-expression of myosin heavy chain (MyHC, green) and β-galactosidase (transfection marker, red) in transfected cells. Cell nuclei were identified by DAPI staining (blue). Scale bar: 12 μm. B. Myogenic differentiation was quantified as the formation of multinucleated cells (≥3 nuclei/ β-galactosidase positive cell), revealing a >3 fold reduction in cell fusion upon Pax7 or Pax7-FKHR co-expression. A and B: MyoD to Pax7/Pax7-FKR molar ratio= 1:2. A and B are representative of at least three independent experiments. Error bars= standard deviation. C. Pax7-FKHR exhibits a less marked inhibitory effect on cell cycle compared to Pax7, measured by BrdU incorporation in asynchronous populations of C3H10T1/2 cells. Mean values are representative of at least two independent experiments. Error bars= standard deviation. *P < 0.001; **P < 0.01. D. Right panel: Pax7-FKHR represses MyoD transcriptional activation (>3 fold reduction) of the 4RTK reporter gene during myogenic conversion of C3H10T1/2 cells. Note that under same conditions, Pax7 represses MyoD activity over 10 fold. MyoD to Pax7/Pax7-FKR molar ratio= 1:2. *, **P < 0.01; # P < 0.05; ## P < 0.01. Left panel: Pax7-FKHR co-expression represses transcriptional activation mediated by the Gal4-MyoD fusion protein in a dose dependent manner. Mean values are representative of at least three independent experiments. Error bars= standard deviation. *P < 0.01; **P < 0.001.