C2C12 myoblasts enter alternate states of arrest.

<p>(<b>A</b>) Asynchronous proliferating myoblasts (MB) were held in suspension in mitogen-rich media to induce quiescence (arrest in an undifferentiated mono-nucleated state; G₀ MB), or shifted to mitogen-poor medium for 96 hrs to induce differentiation (arrest coupled with fusion into multinucleated myotubes; MT). Actin staining (Oregon Green-Phalloidin, top panel) reveals the distinct morphology of these 3 states. Nuclei are detected with Hoechst 33352. DNA synthesis was analyzed by detection of BrdU incorporated in a 15-minute pulse (middle panel)- ∼40% of cycling MB are labeled, while less than 1% of G₀ MB synthesize DNA. In differentiated cultures (MT), only residual mono-nucleated cycling cells (5–10%) incorporate label whereas myotube nuclei do not. Expression of determination factor MyoD can be detected in ∼50% of proliferating MB, is lost in G₀ but sustained in MT (lower panel). (<b>B</b>) FACS analysis of DNA content of cycling (Asynchronous MB) and 48-hr suspension-cultured populations confirms that >97% of cells in suspension (G₀ MB) possess a 2C DNA content while >40% of cells in an asynchronous culture have replicated their genomes (representative graphs from 4 independent experiments). (<b>C</b>) Muscle transcription factor expression distinguishes alternate states of arrest. Q-RT-PCR analysis of specification/survival factor Pax7, determination factor MyoD, and early differentiation marker Myogenin (MyoG) in asynchronous proliferating myoblasts (As), suspension-arrested MB (G₀) and differentiated MT (MT). Values represent normalized fold differences between GAPDH (control) mRNA and myogenic mRNAs in each sample (n = 3). (<b>D</b>) Cell cycle inhibitor expression distinguishes alternate states of arrest-G₀ MB and MT express different combinations of cyclin-dependent kinase inhibitors p21/p27 and Rb-related p130. Immuno-detection of p21, p130 or p27 in MT and G₀; p21 (green) co-localizes with MyoG (red) in all nuclei of differentiated MT but neither factor is expressed in G₀; p130 is specific to G₀ and p27 is expressed in both G₀ and MT. Data depicted is representative of three independent experiments. (<b>E</b>) G₀ arrest is reversible. DNA synthesis in asynchronous MB (Mb), 48-hour suspension cultures (G₀) and G₀ MB replated for 6 or 24 hours (R6, R24). BrdU detected as in Fig. 1A. Values represent mean<u>+</u>SEM (n = 3). (<b>F</b>) MyoD expression is suppressed in G₀ and restored during early reactivation. Asynchronous cultures (A) were arrested in G₀ (48 hrs) and reactivated by replating for 2 to 24 hrs (R2–R24). MyoD expression detected as in Fig. 1A. Values represent mean<u>+</u>SEM (n = 3).