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Figure 2 | Skeletal Muscle

Figure 2

From: Regulation of DMD pathology by an ankyrin-encoded miRNA

Figure 2

Overexpression and inhibition of miR-486 causes profound cellular changes in myoblasts and myotubes. (A) Phase contrast and GFP images of normal human myoblasts overexpressing miR-486, anti-miR-486 and a scrambled miRNA negative control virus are shown. Note that the myoblasts overexpressing the miR-486 inhibitor (anti-miR-486) appear rounded and have not flattened out (inset). (B) Day 4 differentiated normal human myotubes infected with the same lentiviral vectors are shown. Note the aggregate of clumped myotubes (inset) in cells infected with the anti-miR-486 lentivirus. (C) Higher magnification (×40) of normal human myoblasts infected with lentivirus expressing scrambled miRNA, miR-486 and anti-miR-486. The cells show detection of the GFP reporter and 4',6-diamidino-2-phenylindole (DAPI) to detect the DNA. Note that the anti-miR-486-infected myoblasts are rounded (arrowheads) and increased in overall size compared to the scrambled miRNA and miR-486-overexpressing myoblasts. (D) MF20 (red) staining of normal human myoblasts overexpressing scrambled miRNA, miR-486 or anti-miR-486 lentivirus. Chart indicates reduced levels of MF20+ myoblasts that express anti-miR-486 lentivirus; conversely, myoblasts with high levels of miR-486 have increased amounts of MF20+ myoblasts. Ten fields of GFP-positive (not shown) and DAPI-positive cells were counted for the presence of MF20+ staining. Values were normalized to the scrambled miRNA. *P < 0.005 and **P < 0.05. Scale bar indicates 35 μm. (E) FACS histogram reveals an increase in cell size in myoblasts infected with anti-miR-486 lentivirus. The x-axis represents forward scatter height (FSC-H), and the y-axis represents the percentage cell size relative to the control scrambled miRNA myoblasts. FSC-H is quantified in the table that shows that anti-miR-486 (blue line) have increased in cell size compared to myoblasts overexpressing miR-486 (green line) and scrambled miRNA controls (red line). *P = 0.028 ± SEM.

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