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Distinct changes in intranuclear lamin A/C organization during myoblast differentiation

Centre for Cellular and Molecular Biology, Hyderabad 500 007, India

View larger version (37K): [in a new window]   Fig. 1. Colocalization of lamin A with RNA splicing factors. Methanol-fixed C2C12 myoblasts were labeled with mAb LA-2H10 and antibodies to (A) SC-35 or (B) U5-116 kDa. Confocal overlays of the doubly stained cells are shown in the merged panel, where the yellow color highlights structures stained by both antibodies in a single optical section of 0.5 µm. Scale bar: 10 µm.

View larger version (88K): [in a new window]   Fig. 2. Immunolocalization studies with undifferentiated and differentiated muscle cells. C2C12 myoblasts (A) and myotubes (B) were fixed in methanol and labeled with antibodies to lamin A (LA-2H10, LA-2B3), lamin B1 (LB-P), RNA splicing factors (SC-35, U5-116 kDa) or myosin. (C) Unfixed, frozen transverse sections of adult skeletal muscle were stained with LA-2H10 or LA-2B3 (green) and counterstained with DAPI (red). A single myofiber is depicted in each panel. Scale bar: 10 µm.

View larger version (89K): [in a new window]   Fig. 3. Immunoblot analysis of C2C12 cell lysates. Cell lysates were prepared from C2C12 cells kept in differentiation medium for 0-3 days and samples (50 µg protein) were resolved by 10% SDS-PAGE, transferred to nitrocellulose filters and immunoblotted with antibodies to lamins and myosin. A representative gel stained with Coomassie Blue is also shown (CB). Molecular mass markers indicated on the left (from top to bottom) are: phosphorylase b, 94 kDa; albumin 67 kDa; ovalbumin, 43 kDa; and carbonic anhydrase, 30 kDa.

View larger version (41K): [in a new window]   Fig. 4. Localization studies in extracted cells. C2C12 myoblasts (MB) and myotubes (MT) were extracted with detergents, digested with nucleases and treated with 2M NaCl, followed by formaldehyde fixation and labeling with (A) mAb LA-2H10, (B) mAb LA-2B3, (C) mAb SC-35 and (D) m3G antibody to capped RNA, and with DAPI. Unextracted cells are shown as controls. Images of optical sections of 1 µm are displayed. Scale bar: 10 µm.

View larger version (29K): [in a new window]   Fig. 5. Localization of lamin A speckles and markers for myoblast differentiation. (A,B) C2C12 cells were kept in differentiation medium for 0-72 hours, fixed with formaldehyde and labeled with mAb LA-2H10 and myogenin antibody or LA-2H10 and myosin antibody. (C,D) C2C12 cells were maintained in differentiation medium for 6-24 hours, fixed with formaldehyde and labeled with LA-2H10 and myogenin antibody, or LA-2H10 and p21 antibody. (E,F) Quantitative analysis of data in C,D, with n=150 cells at each time point. Arrows in C indicate cells expressing myogenin but not lamin A speckles. Scale bar: 10 µm.

View larger version (49K): [in a new window]   Fig. 6. Localization studies in quiescent cells. (A-C) C2C12 myoblasts that were normally dividing, induced to become quiescent or subsequently reactivated for 24 hours, were fixed and labeled with the indicated antibodies. Similarly, dividing, quiescent and reactivated non-muscle cells (mouse C3H10T1/2 fibroblasts, bottom) were fixed and labeled with mAb LA-2H10. Scale bar: 10 µm.

View larger version (30K): [in a new window]   Fig. 7. Reactivation of quiescent C2C12 cells. (A) Incorporation of BrdU and staining with LA-2H10 were assayed with quiescent C2C12 cells reactivated for 2-24 hours. (B) Quantitative analysis of data in A with n=120 cells at each time point. (C) Quiescent C2C12 cells were extracted with detergent and nucleases as described in Fig. 4 and stained with mAb LA-2H10 and DAPI. Scale bars: 10 µm.