Myogenic fusion efficiency showing percentage of nuclei present within singly nucleated cells, those within cells containing two or three nuclei, and those within cells with four or more nuclei. Dex-treated cells showed a significant increase in myotube formation as evidenced by the marked increase in nuclei present within cells with four or more nuclei over that of untreated cells. A total of 1930 nuclei were counted from untreated cells and 2116 nuclei were counted from Dex-treated C2C12-1 cells three days after switching to differentiation media. Error bars represent standard error. P-values greater that p<0.05 were considered significant.

Western blot analysis of dysferlin and myogenic proteins from the C2C12 time course in the presence or absence of 100nM Dex. Dex treatment increased dysferlin protein levels in C2C12 cells earlier in the time course of differentiation than vehicle control treatment. C2C12-1 cells were cultured with vehicle control (-) or 100nM Dex (+) and protein extracts were prepared from each day of the time course (Days 1 – 7). After harvesting the Day 2 time point, the rest of the cultures were switched from proliferation media to differentiation media for Days 3 – 7. Sixty micrograms of protein were loaded for each time point and the samples were loaded pairwise: minus and plus Dex for each time point. “C” represents mouse muscle control extract. GAPDH protein levels are shown as the loading control. Ratios of pixel intensities in each dysferlin band normalized to the corresponding internal control GAPDH band are shown below the immunoblots.

Myogenic fusion efficiency was decreased in dysferlin-deficient C2C12 cells. Myogenic fusion efficiency was determined three days after the switch to differentiation media (Day 5 of time course). Percentage of DAPI-stained nuclei present within desmin-immunostained singly nucleated cells, those within cells containing two or three nuclei, and those within cells with four or more nuclei are shown in the graph. A. Dysferlin-deficient C2C12 cells (shRNA) showed decreased myogenic fusion of mature myotubes (4 or more nuclei) compared to parental C2C12 P9 cells even with Dex treatment. B. Representative immunofluorescent images from parental C2C12 P9 cells and dysferlin-deficient C2C12 cells in the presence or absence of 100nM Dex are shown in lower panels. Desmin immunostaining appears green and DAPI is blue. A total of 1926 nuclei and 2672 nuclei were counted from vehicle control-treated parental C2C12 P9 and dysferlin-deficient C2C12s, respectively. A total of 1780 nuclei and 2178 nuclei were counted from Dex-treated parental C2C12 P9 and dysferlin-deficient C2C12s, respectively. Error bars represent standard error. P-values greater that p<0.05 were considered significant.

Real time PCR analysis of mRNA levels in C2C12-1 cells growing in the presence or absence of 100nM Dex. A. Dex treatment significantly increased dysferlin mRNA levels in C2C12-1 cells over that of vehicle control treatment on Days 1 – 5 of the time course of differentiation. Differences in mRNA levels were not significant on Days 6 and 7. B. Dex treatment significantly increased myoferlin mRNA levels during proliferation. After the switch to differentiation media, differences in mRNA levels leveled out and were not significantly different between the cultures. C. Relative MyoD mRNA levels. D. Relative myogenin mRNA levels. Based on geNorm analysis of six candidate reference genes, mRNA levels were normalized to the reference genes GAPDH and eEF1ε1. qPCR data are graphed as fold induction relative to Day 1, vehicle control-treated C2C12-1 cells from three assays each run in triplicate. Error bars represent standard error. Differences greater than p < 0.05 are indicated by the * and were considered significant.

Immunofluorescence and confocal microscopy images of dysferlin in C2C12-1 cells treated with vehicle control or 100nM Dex. Dex treatment increased dysferlin levels and enhanced the size of C2C12-1 myotubes compared to vehicle control-treated cells. A. Immunofluorescence microscope images of C2C12-1 on Day 2 of time course. C2C12-1 cells growing in proliferation media with vehicle control (top panels) or 100nM Dex (middle and bottom panels), 100× magnification. B. Confocal microscope images of C2C12-1 cells on Day 6 of time course: vehicle control (top panels), and 100nM Dex (bottom panels), 40×, bar represents 75 μm. Rabbit polyclonal anti-dysferlin antibody was used with Texas Red-conjugated secondary antibody. All samples were counterstained with the nuclear stain, DAPI.

Western blot analysis of dysferlin and myogenic differentiation proteins with GAPDH as a loading control during each day of the differentiation time course (Days 1 – 7) in parental C2C12 P9 cells and dysferlin-deficient C2C12 cells treated with vehicle control (-) or 100nM Dex (+). A. Parental C2C12 cells (P9). B. Dysferlin-deficient C2C12 cells (shRNA).