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Fig. 7. FHL3 overexpression reduces MyoD transcription of luciferase reporter genes. (A) C2C12 cells were co-transfected with various combinations of expression vectors for Vector (HA-vector), HA-FHL3, Gal4DBD and/or Gal4DBD-MyoD, together with a Gal4-luciferase and Renilla luciferase reporters. Transfected myoblasts were maintained in growth media for 48 hours and assayed for luciferase activity. Luciferase values were corrected for background luminescence, normalised to Renilla luciferase activity to adjust for transfection efficiency and standardised relative to Gal4DBD-expressing samples (arbitrarily defined as 1 relative luciferase unit-RLU). Gal4DBD activity is represented by grey bars, Gal4DBD-MyoD by white bars, error bars represent ± s.e.m. (n=6, *P<0.05). Co-transfection with Vector or HA-FHL3 is shown below graph. (B) C2C12 cells were co-transfected as above. Transfected myoblasts were maintained in growth media for 24 hours and switched to differentiation media for 48 hours, and then assayed. Gal4DBD activity is represented by grey bars, Gal4DBD-MyoD by white bars, error bars represent ± s.e.m. (n=7, *P<0.05). Co-transfection with Vector or HA-FHL3 is indicated below figure. (C) Lysates from C2C12 myoblasts transfected with pCMX-Gal4DBD or pCMX-Gal4DBD-MyoD for 24 hours, were immunoblotted to confirm recombinant protein expression. (D) Lysates from C2C12 myoblasts transfected with pCGN or pCGN-FHL3 for 24 hours, were immunoblotted to confirm recombinant protein expression. (E) C2C12 cells were co-transfected with various combinations of expression vectors for Vector, HA-FHL3, Flag-vector and/or MyoD-Flag, with a pGL3-MCK (muscle creatine kinase luciferase reporter) and renilla luciferase. Assays were performed under differentiation conditions as outlined above except values were standardised relative to Flag-vector and Vector coexpressing samples (arbitrarily defined as 1 RLU). Flag-vector activity is represented by grey bars, MyoD-Flag by white bars, error bars represent ± s.e.m. (n=3, *P<0.05, **P<0.01). Co-transfection with Vector or HA-FHL3 is shown below the graph.
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