Table 1.
Frequency of mgRb:Rb−/−:p130−/− embryos recovered.
Figure 1.
Analysis of myogenesis mgRb:Rb−/−:p130−/− double mutant embryos.
(A) Appearance of ctrl, mgRb:Rb−/− and mgRb:Rb−/−:p130−/− embryos at E16.5 (a–c). Hematoxylin & eosin (H&E) histology staining reveals the cellular morphology at mid-sagittal section (d–f), epaxial muscles (g–i) and hypaxial muscles (j–l). Arrowheads point to enlarged nuclei within myofibers. (B) Confocal images showing fast troponin T (green) expression in skeletal muscle sections of control (ctrl) (a–b) mgRb:Rb−/− (c–d) and mgRb:Rb−/−:p130−/− (e–f) embryos. MHC (green) expression in skeletal muscle sections of ctrl (g) mgRb:Rb−/− (h) and mgRb:Rb−/−:p130−/− (i) embryos. DAPI was used to counter-stain nuclei (blue). (C) Top, western blot analysis for pRb and p107 in DM-2 ctrl, mgRb:Rb−/− and mgRb:Rb−/−:p130−/− myoblast cultures. Tubulin was used as loading control. Bottom, western blot analysis of p130 in DM-2 ctrl and mgRb:Rb−/−:p130−/− cultures. Arrow indicates location of p130. Lower band represents a splice variant or cross-reactive protein. (D) Average number of myotubes counted on indicated days post-differentiation. Each time point represents an average ± s.d. of 6 fields at 200X (n = 4).
Figure 2.
Differentiation of mgRb:Rb−/−:p130−/− DKO myoblasts.
(A) Confocal microscopy analysis for BrdU incorporation in ctrl, mgRb:Rb−/− and mgRb:Rb−/−:p130−/− myotubes at DM-2. Myoblasts were differentiated for 1 day, then exposed to 20 µM BrdU for an additional 16 hr in the presence of growth medium (GM) and immuno-stained for MHC (red) and BrdU (green). Arrowheads label BrdU positive nuclei within myotubes. (B) MHC (red) and TUNEL (green) staining at DM-2. Arrowheads indicate TUNEL positive nuclei, which are invariably located outside myotubes. (C) Mitotracker® (red) staining at DM-2. Arrowheads point to large Mitotracker®-positive perinuclear aggregates.
Figure 3.
Rescue of mgRb:Rb−/−:p130−/− myogenic defect by autophagy inhibitors and hypoxia.
(A) Brightfield images of mgRb:Rb−/−:p130−/− myoblasts transduced with Ad.GFP, Ad.Bcl-2 or Ad.RbΔK11 and then induced to differentiate for 14 days. Arrowheads point to myotubes. (B) Average number of mgRb:Rb−/−:p130−/− myotubes following treatment with 3-MA, bezafibrate or DM as indicated. Counts are average ± s.d. of 6 fields at 200X (n = 3). (C) Immunostaining for MHC (green) in ctrl and mgRb:Rb−/−:p130−/− cultures differentiated under normoxia or hypoxia. Note myotubes in mgRb:Rb−/−:p130−/− cultures at DM-5 under hypoxia but not normoxia. Nuclei were counterstained with DAPI.
Figure 4.
BrdU incorporation analysis of RbΔf versus p107−/−:p130−/− myotubes.
(A) Rbf/f myoblasts were transduced with Ad.GFP or Ad.cre and 48 hr later were immunostained for pRb (red). Nuclei were counterstained with DAPI. (B) Rbf/f myoblasts were transduced with Ad.EV or Ad.cre and immunoblotted for pRb 48 hr later. Tubulin served as loading control. (C) Rbf/f myoblasts were transduced with Ad.cre, induced to differentiate for 2 or 6 days and immunostained for MHC (red). (D) Western blot analysis of pRb, p107 and p130 in skeletal muscle of E16.5 Rbf/f:p107+/−:p130+/− (ctrl) and Rbf/f:p107−/−:p130−/− fetuses. (E) Immunostaining for BrdU and MHC in Ad.EV and Ad.cre transduced Rbf/f myoblasts at DM-2. Myoblasts were differentiated for 1 day, then exposed to 20 µM BrdU for an additional 16 hr in the presence of GM and stained for MHC (red) and BrdU (green). Arrowheads label BrdU positive nuclei within myotubes. (F) Immunostaining for BrdU and MHC in Rbf/f, Rbf/f:p107−/−, Rbf/f:p130−/− and Rbf/f:p107−/−:p130−/− myoblasts at DM-2. Myoblasts were differentiated for 1 day, then exposed to 20 µM BrdU for an additional 16 hr in the presence of GM and stained for MHC (red) and BrdU (green). Note absence of BrdU-positive nuclei in myotubes. (G) Quantification of BrdU incorporation in Ad.EV or Ad.cre transduced Rbf/f and Rbf/f:p107−/−:p130−/− myotubes at DM-2.
Figure 5.
Differentiation potential of double and triple KO myoblasts.
(A) Immunostaining for MHC (green) in Ad.EV and Ad.cre transduced Rbf/f, Rbf/f:p107−/−, Rbf/f:p130−/− and Rbf/f:p107−/−:p130−/− myoblast cultures at DM-2. Nuclei were counterstained with DAPI. (B) Quantification of percent multinucleated myotubes relative to total number of MHC-positive cells (myocytes plus myotubes) in Ad.EV and Ad.cre transduced Rbf/f, Rbf/f:p107−/−, Rbf/f:p130−/− and Rbf/f:p107−/−:p130−/− myoblasts at DM-2 under normoxia. Numbers within bars indicate % for the respective samples.
Figure 6.
Increased apoptosis associated with differentiation of double and triple KO myoblasts.
(A) Mitotracker® staining of Ad.EV and Ad.cre transduced Rbf/f, Rbf/f:p107−/−, Rbf/f:p130−/− and Rbf/f:p107−/−:p130−/− myoblasts at DM-2. Arrowheads point to large perinuclear aggregates in Ad.cre transduced myotubes. (B) TUNEL staining (green) of Ad.EV and Ad.cre transduced Rbf/f (a–b), Rbf/f:p107−/− (c–d), Rbf/f:p130−/− (e–f) and Rbf/f:p107−/−:p130−/− (g–h) cultures at DM-2. Nuclei were counterstained with DAPI. Note that TUNEL positive nuclei are outside myotubes. (C) Percent increase in TUNEL-positive cells in Ad.cre relative to Ad.EV transduced cultures. Error bars represent s.d. *-p<0.05 and **-p<0.07 t-test comparisons relative to Rbf/f.
Figure 7.
Differentiation of double and triple KO myoblasts under hypoxia.
(A) Immunostaining for MHC (green) of Ad.EV (a,c,e,g) or Ad.cre (b,d,f,h) transduced Rbf/f, Rbf/f:p107−/−, Rbf/f:p130−/− and Rbf/f:p107−/−:p130−/− cultures at DM-5 in hypoxia. Inlets, DAPI staining for nuclei. (B) Quantification of myotube formation in Rbf/f, Rbf/f:p107−/−, Rbf/f:p130−/− and Rbf/f:p107−/−:p130−/− cultures transduced with Ad.EV or Ad.cre and induced to differentiate 48 hr later for 5 days. Counts represent the average number of myotubes at DM-5 of 6 representative fields (n = 4); error bars represent s.d. *-p<0.05 and **-p<0.07. (C) Quantification of percent multinucleated myotubes relative to total number of MHC-positive cells in Ad.EV or Ad.cre transduced Rbf/f, Rbf/f:p107−/−, Rbf/f:p130−/− and Rbf/f:p107−/−:p130−/− cultures at DM-5 under hypoxia. Numbers within bars indicate % for respective samples. (D) Quantification of myotube formation in Rbf/f, Rbf/f:p107−/−, Rbf/f:p130−/− and Rbf/f:p107−/−:p130−/− cultures transduced with Ad.EV or Ad.cre and induced to differentiate in hypoxia. Counts were conducted at DM-2 and DM-6. Error bars represent s.d.
Figure 8.
Evidence that bi-nuclear TKO myotubes originate from nuclear duplication, not cell fusion.
(A) Bright-field images of Ad.EV and Ad.cre transduced Rbf/f:p107−/−:p130−/− cultures at DM-6 in hypoxia. Arrowheads point to myotubes. Note the presence of a thin myotube in Ad.cre transduced culture. (B) Top row, low magnification image of Ad.EV or Ad.cre transduced Rbf/f:p107−/−:p130−/− cultures immunostained for pRb (green) at DM-2 (400x). Bottom row, high magnification (630x) of Ad.EV or Ad.cre transduced Rbf/f:p107−/−:p130−/− cultures induced to differentiate and then immunostained for pRb (green), demonstrating absence of detectable pRb within binuclear myocyte. Nuclei were counterstained with DAPI. Arrowheads point to nuclei in the myocyte. (C) Immunostaining for MHC (red) and BrdU (green) at DM-2 of Ad.EV and Ad.cre transduced Rbf/f:p107−/−:p130−/− cultures, which were induced differentiate after equal mixing of BrdU+ labeled and BrdU− myoblast populations. Note mixed BrdU+ and BrdU− nuclei in control myotube (top panel) but only BrdU−:BrdU− or BrdU+:BrdU+ nuclei in TKO myotubes (middle and bottom, respectively).