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. 2010 Oct 18;191(2):291–301. doi: 10.1083/jcb.201005067

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© 2010 Ciavarra and Zacksenhaus

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 1. — Rb^−/− myoblasts transiently differentiate to form short myotubes that do not twitch, express autophagy markers, and slowly degenerate. (A) Western blot analysis for pRb in DM-2 cultures. (B) Immunostaining for MHC in DM-3 and DM-6 differentiating myotubes. Nuclei were stained with DAPI (blue). (C) Mean number of myotubes on the indicated days after differentiation. Each time point is mean ± SD of six fields from six independent experiments. (D) TUNEL staining. Arrowheads indicate TUNEL-positive nuclei (green). (E) LC3-RFP expression in control (ctrl) and Rb^−/− myotubes. Insets show nuclear DAPI stain (blue). (F) Representative Western blot (n = 3) for LC3. Chloroquine was added 12 h before cell harvest. (G) Representative Western blot for LC3 in E16.5 control and mgRb:Rb^−/− muscles. (H) Quantification of LC3-II/tubulin ratio in E16.5 mgRb:Rb^−/− skeletal muscles relative to control as depicted in G (mean ± SD; n = 3). *, P = 0.016 by Student’s t test. (I) Representative Western blot for LC3-I and LC3-II expression in whole cell lysates from the indicated myotubes transduced with Ad.GFP or Ad.RbΔK11. (J) Quantification of LC3-II/tubulin ratio by Western blots as shown in I. Data represent mean ± SD (n = 7). *, P = 0.047; **, P = 0.005 by analysis of variance.