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Figure 8


Fig. 8. siRNA-mediated FHL3 knockdown increases MyoD transcription of luciferase reporter genes. (A) C2C12 cells were co-transfected with various combinations of expression vectors for Gal4DBD or Gal4DBD-MyoD and siRNA oligonucleotides as indicated, with a Gal4-luciferase and Renilla luciferase reporters. Assays were performed under differentiation conditions. Gal4DBD activity is represented by grey bars, Gal4DBD-MyoD by white bars, error bars represent ± s.e.m. (n=4 *P<0.05, **P<0.01). Co-transfection with specific siRNAs is shown below the graph. (B) Cellular extracts (20 µl) prepared from C2C12 cells transfected with the indicated constructs and used for the luciferase assay shown in A), were also immunoblotted with the anti-FHL3 antibody to confirm siRNA-mediated FHL3 knockdown and with total actin antibody as a protein loading control. (C) C2C12 cells were co-transfected with various combinations of expression vectors for Flag-tagged vector or MyoD-Flag and siRNA oligonucleotides as indicated with a pGL3-MCK and Renilla luciferase reporters. Assays were performed under differentiation conditions. Flag-tagged vector activity is represented by grey bars, MyoD-Flag by white bars, error bars represent ± s.e.m. (n=8, *P<0.05, ***P<0.001). Co-transfection with specific siRNAs is shown below the graph. (D) Cellular extracts (20 µl) prepared from C2C12 cells transfected with the indicated constructs and used for the luciferase assay shown in C), were also immunoblotted with the anti-FHL3 antibody to confirm siRNA-mediated knockdown of FHL3 protein and with total actin antibody as a protein loading control.





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