Fig. 1. FHL3 overexpression retards C2C12 cell differentiation. (A) C2C12 myoblast cell lines that stably express hemagglutinin (HA) vector [Vector (1) and Vector (2)] or HA-FHL3 [HA-FHL3 (1) and HA-FHL3 (2)] were generated in duplicate. Lysates were immunoblotted for FHL3 or total (pan) actin. Immunoblots are representative of four independent experiments. (B) The mean level of FHL3 protein expression in cell lines stably expressing HA-FHL3 versus those expressing Vector was quantified by densitometry and standardised to total actin (n=4, ^*P<0.05, ^**P<0.01). FHL3 protein levels standardised to actin were expressed relative to vector levels that were arbitrarily set at 1. (C) Lysates from differentiating C2C12 myoblasts stably expressing vector or HA-FHL3 were immunoblotted using anti-HA or anti-actin antibodies. Immunoblots are representative of three independent experiments. (D) C2C12 myoblasts stably expressing vector or HA-FHL3 were differentiated, stained with anti-MHC antibodies (green) and propidium iodide (red), and then imaged by confocal microscopy. Bar, 40 µm. Shown are representative images following 120 hours of differentiation. Immunofluorescence images are representative of three experiments for all cell lines. (E) Cells were co-stained with anti-MHC antibodies and propidium iodide. The differentiation index was determined by scoring the proportion of nuclei, of the total number of nuclei within MHC-positive cells (myocytes and myotubes). A minimum of 116 nuclei were scored per stable cell line per time point for each of three independent experiments (^*P<0.05). Dashed line represents combined results of Vector (1) and (2) lines and black line shows combined results from HA-FHL3 (1) and (2) cell lines. (F) Cells were co-stained with anti-MHC antibodies and propidium iodide. The degree of cell fusion was quantified by determining the average number of nuclei per MHC-positive cell. A minimum of 42 and 69 MHC-positive cells were scored following 96 and 120 hours differentiation respectively, for each of three independent experiments, (^*P<0.05, ^**P<0.01). Dashed line shows combined results of Vector (1) and (2) lines, and black line represents HA-FHL3 (1) and (2) cell lines combined results. Error bars represent ± s.e.m.