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Fig. 3. FHL3 siRNA-knockdown accelerates C2C12 cell differentiation. C2C12 myoblasts were transfected with siRNA oligonucleotides, grown for 48 hours and then differentiated for up to 72 hours. In B,D,E, control siRNA-transfected cells are represented by dashed lines, siRNA FHL3 #2 by black solid lines and siRNA FHL3 #3 by grey solid lines, error bars represent ± s.e.m. *P<0.05, ***P<0.001. (A) Cell lysates from siRNA transfected C2C12 cells differentiated for up to 72 hours were immunoblotted for FHL3 or total actin. (B) FHL3 protein levels were quantified by densitometry and standardised to total actin (n=3). (C) siRNA-transfected C2C12 cells were differentiated, stained with anti-MHC antibodies (green) and propidium iodide (red) and imaged by confocal microscopy. Bars, 40 µm. Shown are representative images following 72 hours of differentiation. (D) The differentiation index was quantified using a minimum of 320 nuclei scored per transfected siRNA oligonucleotide species, per time point, for each of four independent experiments. (E) The average number of nuclei per MHC-positive cell was determined for siRNA-transfected cells, a minimum of 56 and 40 MHC-positive cells were scored following 48 and 72 hours differentiation, respectively, per transfected siRNA oligonucleotide species (n=4).
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