FoxO1 shuttles between nucleus and cytoplasm during myogenic differentiation.

<p>The subcellular localization of FoxO1 in C2C12-FoxO1-wt (<b>A</b>)<b>,</b> control (<b>C</b>)<b>,</b> and C2C12-FoxO1-AAA (<b>D</b>) cells at proliferating (PMB), confluent (CMB), and myotube (MT) stages were detected with immunofluorescence microscopy. The relative distribution of FoxO1 in C2C12-FoxO1-wt cells at these stages was shown in (<b>B</b>). The signals of FoxO1 and MHC were detected with Alexa fluor 488 (green) and 486 (red), respectively, conjugated secondary antibody, and nucleus was stained with DAPI (blue). Arrows at the bottom panels point to myotubes. ud: undectable. The original magnification is 200X. (<b>E</b>) Vectors expressing GFP-FoxO1-wt (top panel) or GFP-FoxO1-AAA (bottom panel) were transfected into parental C2C12 cells of myotube stage (4 days in DM) and the GFP signal in multinucleated myotubes (detected by MHC antibody, red) was viewed 24 hr after transfection. (<b>F</b>)<b>,</b> and (<b>G</b>)<b>,</b> nuclear shuttling of FoxO1 is suspended in myotubes. Proliferating myoblasts (<b>F</b>) and differentiated myotubes (<b>G</b>) of C2C12-FoxO1-wt were treated with vehicle or leptomycin B (LPB, 2 ng/ml) for 24 h before fixed and stained for FoxO1 and MHC as described above. The original images of (E), (F) and (G) were taken at 400X magnification.