Article |
Address correspondence to Michael A. Rudnicki, Molecular Medicine Program, Ottawa Health Research Institute, 501 Smyth Rd., Ottawa, Ontario, Canada, K1H 8L6. Tel.: (613) 739-6740. Fax: (613) 737-8803. email: mrudnicki{at}ohri.ca
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Abstract |
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Key Words: pRb; primary myoblasts; proliferation; MyoD; myogenin
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Introduction |
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MRFs are subject to regulation that acts to couple MRF activity to the cell cycle. Hypophosphorylated pRb was suggested to bind MyoD and this association to be required for MyoD-mediated activation of E-boxcontaining muscle-specific promoters (Gu et al., 1993). However, direct binding between pRb and MyoD has been ruled out by in vivo and in vitro assays (Zhang et al., 1999a,b). Therefore, pRb likely potentiates MyoD activity via an indirect mechanism not involving binding Rb to MyoD.
Proliferating myoblasts express Id, activated Cdk4, low levels of hyperphosphorylated Rb, and free E2Fs as well as E2Fs complexed largely with p107. Activated Cdks and Id both stimulate cell cycle progression. In contrast, myotubes express high levels of p21 and hypophosphorylated pRb. Despite the abundance of hypophosphorylated pRb, p130E2F4 complexes are the predominant E2F complexes in the myotube (Corbeil et al., 1995). Terminal differentiation and protection against apoptosis is maintained by Cdk inhibitors (p21, p27, etc.) and high expression of hypophosphorylated pRb (Jiang et al., 2000; Peschiaroli et al., 2002; Ho et al., 2004). Together, these data suggest that during myogenic differentiation pRb plays a role distinct from the conventional repression of E2F transcriptional activity.
Newborn mice lacking pRb exhibit multiple deficits including severe deficiencies in the formation of skeletal muscle (Zacksenhaus et al., 1996; de Bruin et al., 2003; Wu et al., 2003). Studies using MyoD-converted pRb-deficient embryonic fibroblasts have suggested that Rb is essential for both MyoD and MEF2 transcriptional activity, as well as maintaining the terminally differentiated state (Schneider et al., 1994; Novitch et al., 1996, 1999). Although pRb-deficient fibroblasts transfected with MyoD become myogenic and express early muscle markers such as myogenin, expression of late markers such as myosin heavy chain (MHC) is reduced. In addition, serum restimulation of these differentiated pRb-deficient myoblasts results in BrdU incorporation and, thus, S-phase entry and DNA synthesis. However, these cells are unable to enter mitosis. Moreover, forced expression of MyoD in a variety of Rb/ fibroblastic cells results in apoptosis that appears to be p21 dependent (Peschiaroli et al., 2002). In the absence of N-ras, pRb-deficient embryos exhibit normal muscle differentiation without apoptosis, suggesting a role for signaling downstream of N-ras in provoking cell death in Rb/ muscle (Takahashi et al., 2003).
Rb plays a key role in controlling cell cycle progression through the G1 restriction point for entry into S-phase (Stevaux and Dyson, 2002). During myogenic differentiation, proliferating myoblasts must also exit the cell cycle from the G1 phase, before the restriction point (Perry and Rudnick, 2000). Therefore, it can be hypothesized that pRb plays an analogous role in myoblasts by regulating the switch from proliferation to differentiation.
To investigate the requirement for pRb in myogenic differentiation, we examined the proliferation and differentiation potential of primary myoblasts in which a floxed Rb allele was deleted either before or after differentiation. Our experiments unequivocally establish that pRb is required for progression of the differentiation program and not for maintenance of the differentiated state.
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Results |
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Rbf/f males were crossed with Rbf/wt:Myf5-Cre females to generate Rbf/f:Myf5-Cre progeny. Notably, no viable Rbf/f:Myf5-Cre mice were identified after genotyping over 95 offspring. Examination of newborn litters revealed the expected Mendelian proportion of Rbf/f:Myf5-Cre pups. However, the newborn pups lacking pRb in myoblasts were motionless, became cyanotic, and failed to survive. Therefore, we concluded that Rbf/f:Myf5-Cre mice exhibited a phenotype similar to that of other Rb knockout mouse models (Lasorella et al., 2000; de Bruin et al., 2003).
Histological examination of skeletal muscle revealed the presence of severe differentiation deficits (Fig. 1, AF). Hind limb muscles exhibited a dramatic reduction in mass with a complete absence of mature fibers compared with littermate controls (n = 3 independent animals; Fig. 1, compare A with B). In addition, the morphology of the residual muscle fibers in the Rbf/f:Myf5-Cre mice was short and irregular in shape (Fig. 1 D). Moreover, the long and orderly parallel arrangement of the fibers typically seen in the wild-type controls was absent in the Rbf/f:Myf5-Cre muscle (Fig. 1, compare C with D). These results confirm the well-established requirement for pRb in myogenesis.
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Adenoviral infection and expression of Cre resulted in the complete excision of the floxed region of Rb as assessed by 32P end-labeled PCR genotyping and Western blot analysis (Fig. 4, A and B). Our method of 32P end-labeled PCR genotyping was capable of detecting an Rbflox:Rbexcised allele ratio as low as 104 when amplified from 20 ng of total DNA (unpublished data). Moreover, pRb protein was below the level of detection by Western blot analysis in Ad-Creinfected Rbf/f myoblasts (n = 3 independent isolates and more than three infections per isolate). Additionally, pRb protein was undetectable by immunofluorescence in the nuclei of the Ad-Creinfected myoblasts after 2 d in differentiation media (DM), when pRb is normally expressed at high levels (n = 3; Fig. 4 C). Ad-Creinfected myoblasts appeared to be smaller and more compact than the control-infected cells. Our visual assessment was validated by flow cytometry analysis that confirmed the actual decrease in average size of pRb-deficient myoblasts relative to controls (unpublished data).
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Rb-deficient primary myoblasts are incapable of differentiation
Primary myoblasts can be quickly and efficiently induced to exit the cell cycle and initiate the differentiation program by exposure to low serum conditions. To investigate the differentiation potential of primary myoblasts lacking pRb, we exposed Ad-Creinfected Rbf/f myoblasts to low serum conditions using standard procedures (Sabourin et al., 1999). We observed a rapid loss of cell viability (Fig. 5) together with an inability of the remaining cells to form multinucleated myotubes (see Fig. 6, compare C with F).
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Interestingly, the small number of cells that did survive the low serum conditions appeared incapable of differentiation. The surviving pRb-deficient primary cells were adherent and formed elongated mononuclear myocytes that failed to fuse with closely neighboring myocytes to form multinucleated myotubes (Fig. 4 C, bottom left).
We conducted immunofluorescent staining experiments to examine the cellular expression and localization patterns of MyoD, myogenin, and MHC (n = 3 independent isolations and infections; Fig. 6, AL). MyoD is expressed in proliferating myoblasts and is down-regulated during differentiation. Myogenin is not expressed in proliferating myoblasts and is up-regulated in mononuclear cells as an early response gene during induction of differentiation. Finally, MHC is up-regulated relatively late in the differentiation program and is typically detected in multinucleated myotubes (Sabourin et al., 1999).
Under growth conditions, MyoD was localized to the nuclear compartment and similarly expressed in almost all of the pRb-deficient and wild-type primary myoblasts (Fig. 6, A and D). In addition, high levels of MyoD were detected in the nuclei of the pRb-deficient and wild-type myoblasts by day 1 of differentiation (Fig. 6, B and E). After differentiation, MyoD protein was down-regulated to low levels in virtually all the nuclei of wild-type multinucleated myotubes (Fig. 6 C). In contrast, 36% of the pRb-deficient Rbf/f:Ad-Cre myocytes continued to express high levels of MyoD (Fig. 6 F). Western blot analysis showed that both wild-type and mutant myoblasts expressed similar levels of MyoD in subconfluent growth conditions (Fig. 7 C). However, by days 3 and 5 of differentiation, mutant cells had failed to down-regulate MyoD (n = 3; Fig. 7 C).
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After 1 d of differentiation, cytoplasmic MHC expression was detected by immunofluorescence in newly formed wild-type myotubes, whereas little such expression was detected in mutant cells (Fig. 6, compare B with E). By day 5 of differentiation, robust cytoplasmic MHC staining was evident in wild-type multinucleated myotubes (Fig. 6 C). In contrast, the pRb-deficient cells were incapable of completing the differentiation program, as evidenced by the absence of multinucleated myotubes expressing MHC (Fig. 6 F). Western blot analysis confirmed the very low level expression of MHC in Rbf/f:Ad-Crederived cells (Fig. 7 C). Together, these data suggest that the perinatal death of Rbf/f:Myf5-Cre mice is due to an intrinsic defect in differentiation of the myogenic progenitors.
Pax7 is typically expressed at high levels in proliferating myoblasts and is rapidly down-regulated upon induction of differentiation (Seale et al., 2000). Western blot analysis revealed that pRb-deficient primary myoblasts exposed to differentiation medium fail to down-regulate Pax7 (Fig. 7 C). Together, these data suggest that the low numbers of surviving pRb null myoblasts are able to initiate differentiation, but subsequently fail to properly regulate the progression of the myogenic program required for the completion of myogenic differentiation.
Primary myoblasts lacking pRb fail to arrest upon induction of differentiation
To elucidate the cell cycle and differentiation phenotype of the Ad-Creinfected Rbf/f myoblasts, we performed a series of RNA and protein expression analyses for cell cycle and differentiation markers. RNA expression levels of cyclins and cdk's were analyzed under growth conditions and serum withdrawal differentiation conditions (n = 3). Because of RNase protection, pRb-deficient primary myoblasts fail to down-regulate cyclinA2, cyclinB1, cdk1, cdk2, and cdk4 mRNAs in response to serum withdrawal (Fig. 7, A and B).
Mutant pRb-deficient cells displayed increased levels of p107 protein under growth conditions in comparison with the wild-type cells (Fig. 7 C). In addition, postdifferentiation mutant cells continued to express high levels of p107 protein relative to wild-type myotubes (n = 3; Fig. 7 C). However, the levels and pattern of p130 expression appeared normal (n = 3; Fig. 7 C). Therefore, pRb-deficient primary myoblasts display a similar cell cycle phenotype to that of MyoD-transfected pRb-deficient fibroblasts (Schneider et al., 1994; Novitch et al., 1996, 1999).
To investigate whether pRb-deficient myoblasts were appropriately withdrawing from the cell cycle upon induction of differentiation, we performed BrdU incorporation experiments on newly differentiated cultures. Wild-type cells displayed no incorporation of BrdU in cultures exposed for 1 h to BrdU, after 4 d of differentiation (Fig. 8 A). In contrast, 25% of Rbf/f:Ad-Cre myoblasts present after a 4-d exposure to differentiation medium incorporated BrdU (Fig. 8 C). In addition, Rbf/f:Ad-Cre myocytes also resumed cell division and exhibited an
50% increase in total cell numbers after reexposure to growth medium for 24 h (Fig. 8 D). These data are consistent with the hypothesis that Rb-deficient myoblasts display an intrinsic failure of the G1 restriction point necessary for the switch to terminal myogenic differentiation.
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The levels of pRb over 4 d of differentiation were examined by Western blot analysis to allow us to determine the temporal kinetics of pRb elimination by the MCK-Cre transgene (n = 3; Fig. 10 B). Levels of pRb in wild-type and mutant cells were similar in growth media and after 2 d in DM (Fig. 10, compare A with B). However, MCK-Cremediated deletion of the Rbf/f allele resulted in the complete ablation of pRb after 4 d of differentiation (Fig. 10, compare A with B). Interestingly, this late-stage elimination of pRb did not result in the up-regulation of p107 as observed in Rbf/f:Ad-Cre cultures (compare Fig. 10 B with Fig. 7 C). Consistent with the normal differentiation of Rbf/f:MCK-Cre primary myoblasts, MyoD and myogenin levels were subject to normal modulation (Fig. 10 B). Thus, functional pRb is critically involved in the cascade of regulatory events required for progression through myogenic differentiation, but pRb is not required for the maintenance of the terminally differentiated state.
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Primary myoblasts were cultured in DM for 4 d and either BrdU pulsed for 1 h before fixation or stimulated with growth media containing 20% FCS and supplemented with BrdU for 24 h before fixation (Fig. 8). Our experiments revealed that stimulation of Rbf/f:MCK-Cre myotubes for 24 h with growth media did not result in any myotube nuclei entering S-phase, as evidenced by an absence of BrdU incorporation (Fig. 8 F). Moreover, serum stimulation did not result in up-regulation of MyoD and cyclin E proteins in Rbf/f:MCK-Cre myotubes (Fig. 10 C). However, levels of p107 were reinduced in the Rbf/f:MCK-Cre myotubes to approximately half the levels present in the Rbf/f:Ad-Cre myocytes (Fig. 10 C). Additionally, higher levels of cyclin E protein and an activated form of cdk2 were also present in the restimulated Rbf/f:Ad-Cre myocytes but not in the Rbf/f:Ad-Lac-Z and Rbf/f:MCK-Cre myotubes (Fig. 10 C). Therefore, these experiments indicate that pRb is not required to maintain the terminally differentiated state in myotubes that are derived from primary myoblasts.
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Discussion |
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Recent studies have demonstrated that many of the developmental defects observed in the initial Rb knockout studies were primarily caused by a pRb-dependent extraembryonic defect in placental development (Wu et al., 2003). Functional rescue of placental defects by the generation of aggregation chimeras with wild-type tetraploid donor embryos results in Rb/ pups that survive to birth without neuronal or erythroid phenotypes. Notably, the newborn Rb/ pups exhibited ectopic S-phases and apoptosis in the lens, together with extensive deficiencies in skeletal muscle differentiation (de Bruin et al., 2003). The muscle phenotype in these animals is strikingly reminiscent of the Rbf/f:Myf5-Cre muscle phenotype. Importantly, our experiments provide the formal genetic proof that pRb is required in a cell-autonomous manner for skeletal muscle development.
Ad-Creinfected Rbf/f primary myoblasts could not properly form multinucleated MHC-expressing myotubes (Fig. 6). Additionally, Rbf/f:Ad-Cre myoblasts were unable to down-regulate cyclins, cdk's, p107, Pax7, MyoD, and myogenin when withdrawn from serum (Fig. 7). However, upon induction of differentiation, Rbf/f:Ad-Cre myoblasts up-regulated myogenin expression, which is an immediate early marker of commitment for differentiation (Bergstrom et al., 2002). Therefore, we conclude that Rbf/f:Ad-Cre myoblasts enter the differentiation program but fail to progress, as evidenced by continued Pax7 expression, cell division, and failure to form multinucleated myotubes.
Activation of myogenin transcription is directly mediated by MyoD binding to E-boxes located in the proximal myogenin promoter in response to differentiation-inducing signals (Gerber et al., 1997; Bergstrom et al., 2002). Therefore, these data are consistent with the suggestion that myogenin induction occurs independently of Rb transcriptional induction during myogenic differentiation.
During muscle differentiation, pRb becomes hypophosphorylated and mRNA and protein levels increase 10-fold (Martelli et al., 1994; Corbeil et al., 1995). MyoD activation during differentiation is responsible for the transcriptional activation of the Rb (Martelli et al., 1994). More recent studies demonstrate that MyoD activation of Rb transcription requires a cyclic AMPresponsive element in the Rb promoter, which binds CREB protein (Magenta et al., 2003). Moreover, CREB is phosphorylated during myogenic differentiation and recruits MyoD, p300, and pCAF to the Rb promoter (Magenta et al., 2003). Therefore, both Rb and myogenin are immediate early targets of MyoD-mediated transcriptional activation during the switch to myogenic differentiation. Our experiments demonstrate that pRb activity is absolutely required for MyoD-mediated cell cycle exit and completion of the differentiation program, but not for activation of myogenin.
The severe myogenic differentiation defect seen in the Rbf/f:Myf5-Cre mice (Figs. 1 and 2) and the Rbf/f:Ad-Cre primary myoblasts (Figs. 57) was in stark contrast with the normal phenotype observed in the Rbf/f:MCK-Cre skeletal muscle from which pRb was eliminated after the completion of differentiation (Figs. 3 and 9). Furthermore, primary myoblasts isolated from Rbf/f:MCK-Cre mice were also able to properly differentiate into multinucleated MHC-positive myotubes. The Rbf/f:MCK-Cre myotubes lacking pRb did not incorporate BrdU when restimulated with growth media for 24 h (Fig. 8 F). However, Rbf/f:Ad-Cre myocytes were fully capable of incorporating BrdU and resuming cell division upon growth media restimulation (Fig. 8 D). The persistence of activated cdk2 (Fig. 10 C) and an abundance of its regulatory subunit cyclin E (Fig. 10 C) observed in the restimulated Rbf/f:Ad-Cre myocytes, but not in the Rbf/f:Ad-Lac-Z and Rbf/f:MCK-Cre myotubes, are indicative of the actively proliferating state of the restimulated Rbf/f:Ad-Cre myocytes (Cenciarelli et al., 1999). Interestingly, serum restimulation of Rbf/f:MCK-Cre myotubes results in the induction of p107 protein (Fig. 10 C). However, despite the induction of p107 in differentiated myotubes, the cell cycle continues to be checked. Up-regulation of cell cycle genes has previously been reported in serum-treated terminally differentiated C2C12 myotubes. Serum restimulation of C2C12 myotubes induces immediate early genes such as c-fos, c-jun, c-myc, and Id-1 (Tiainen et al., 1996a). Moreover, cell cycle genes such as cyclin D1 and cdc2 were similarly serum-induced to levels comparable to that of proliferation (Jahn et al., 1994), yet no evidence of cell cycle reentry was ever observed. Therefore, Rbf/f:MCK-Cre myotubes are capable of inhibiting DNA synthesis and maintaining the terminally differentiated state in the absence of pRb.
The maintenance of the terminally differentiated state is likely the effect of multiple mechanisms that may include p130E2F4 complexes (Corbeil et al., 1995; Takahashi et al., 2000), cyclin D3 complexes (Cenciarelli et al., 1999), p21 activity (Jiang et al., 2000; Mal et al., 2000), and inhibition of cyclin D1 expression (Skapek et al., 1995; Latella et al., 2001). Notably, forced expression of E2F1 in differentiated C2C12 myotubes is insufficient to induce ectopic DNA synthesis (Tiainen et al., 1996a). However, the expression of viral oncoproteins such as E1A (Tiainen et al., 1996b) and SV40 large T antigen (Gu et al., 1993) in C2C12 myotubes can induce ectopic DNA synthesis, but these oncoproteins are both known to inactivate multiple checkpoint mechanisms (Helt and Galloway, 2003). Therefore, terminal differentiation is a very stable state that appears tolerant of inactivation of single pathways.
A high proportion of pRb-deficient myoblasts undergo apoptosis in response to induction of differentiation. Interestingly, attenuation of N-ras signaling has been suggested to rescue this apoptotic response of Rb mutant myoblasts (Takahashi et al., 2003). The set of genes activated by mitogens via the RasMAPK pathway are very similar to the genes activated after E2F induction (Muller et al., 2001). Moreover, forced expression of E2Fs results in high rates of apoptosis (Stevaux and Dyson, 2002), and elevated rates of apoptosis in the cortex of Rb/ embryos are attenuated by crossing into E2F1 and E2F3 mutant backgrounds (Ziebold et al., 2001). Several proapoptotic genes including caspase 3 have been suggested to represent E2F target genes (Muller et al., 2001). Notably, caspase 3 is up-regulated upon myogenic differentiation and is required for normal progression of the differentiation program (Fernando et al., 2002). Therefore, it is interesting to speculate that pRb may play a protective role in preventing an apoptotic response to activated caspase 3 during myogenic differentiation.
In this paper, we have demonstrated that pRb is essential for progression to terminal myogenic differentiation. Several pRb-dependent mechanisms have been suggested to play important roles in the regulation of myogenesis. Dominant negative N-ras enhances MyoD-mediated expression from an MCK reporter construct in Rb/ fibroblasts (Lee et al., 1999). Additionally, N-ras/:Rb/ compound mutant muscle appears to be relatively normal, suggesting that N-ras plays a role in mediating the defect in differentiation of pRb-deficient myoblasts (Takahashi et al., 2003). Other studies have suggested that dynamic interactions between pRb, HDAC1, and MyoD play an important role (Puri et al., 2001). Alternatively, pRb inhibition of Id2 has been suggested to potentiate MyoD activity (Lasorella et al., 2000). Clearly, the importance of these interactions must be reassessed under a unified context in light of our findings.
We have demonstrated that pRb is required during the early stages of differentiation in order to properly control cell cycle exit and regulate the progression of the differentiation program. However, maintenance of myogenic terminal differentiation does not require pRb. Identifying the early pRb-dependent downstream regulatory network could potentially be valuable in developing highly efficient stem cell therapeutics for a broad range of myopathies. Future studies will address the mechanisms and the downstream targets of pRb regulation during the early stages of skeletal muscle differentiation.
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Materials and methods |
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Single fiber isolation
A single-fiber isolation procedure was performed on the extensor digitorum longus muscle of 8-wk-old Rbf/f:MCK-Cre and Rbf/wt control littermates as described previously (Rosenblatt et al., 1995). 20 fibers were lysed in DNA lysis buffer (100 mM Tris-HCl, pH 8.5, 5 mM EDTA, 0.2% SDS, 200 mM NaCl, and 100 µg/ml proteinase K), and the DNA was precipitated by isopropanol and washed once in 70% ethanol. 200 fibers were lysed in protein extraction buffer (50 mM Tris-HCl, pH 7.4, 0.1% Triton X-100, 5 mM EDTA, 250 mM NaCl, and 50 mM NaF) supplemented with protease inhibitors (0.1 mM Na3VO4, 1 mM PMSF, and 10 µg/ml leupeptin).
32P End-labeled PCR and densitometry analysis
Rb flox forward primer was end labeled with -[32P]ATP (Amersham Biosciences) by T4 kinase (Invitrogen) in a final reaction volume of 20 µl. Rb flox PCR was performed under standard conditions using 0.4 µl of end-labeled primer in each 10-µl PCR reaction. 20 ng DNA was used as a starting template in each PCR reaction. Radiolabeled PCR products were resolved on a 7% denaturing acrylamide gel and exposed on film. Band densities were quantified using ImageJ analysis software. Rbflox:Rbexcised allele ratios were determined by densitometry analysis, and an allele ratio standard curve was constructed. Sensitivity of detection was determined by decreasing the total ratio of Rbflox DNA to Rbexcised DNA in 10-fold steps.
BrdU-pulsed cell cycle analysis
Exponentially growing asynchronous Ad-Cre and Ad-Lac-Zinfected Rbf/f primary myoblasts were pulsed with BrdU for 20 min at a concentration of 30 µM. Myoblasts were trypsinized, pelleted, and washed twice in ice-cold PBS. Myoblasts were stained for BrdU incorporation and total DNA content using the BrdU Flow Kit (BD Biosciences) according to the manufacturer's instructions. Flow cytometry was performed on a FACStar (Beckman Coulter).
TUNEL assay
Ad-Cre and Ad-Lac-Zinfected Rbf/f myoblasts were grown on collagen-coated 4-well poly-L-lysine chamber slides (Lab-Tek). Cells were fixed with 4% formaldehyde in PBS at the 6-h differentiation time point. TUNEL assays were performed using the ApoAlert DNA Fragmentation Kit (CLONTECH Laboratories, Inc.) according to the manufacturer's instructions. TUNEL staining was analyzed and photographed on an Axioskop microscope (Carl Zeiss MicroImaging, Inc.) equipped with a UV source and FITC, rhodamine, and HOECHST detection filters.
RNase protection and Western analysis
Total RNA was extracted from Ad-Cre and Ad-Lac-Z Rbf/f primary myoblasts using the RNeasy Mini-Kit (QIAGEN). RNase protection assay was performed using the RiboQuant kit along with the multi-probe template sets m-CYC-1 and m-CC-1 (BD Biosciences). 2 µg of total RNA from each time point was used for the ribo-probe hybridization incubation. The RNase protection assay was completed according to the manufacturer's instructions.
Primary myoblasts were harvested and lysed in RIPA extraction buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 5 mM EDTA, 150 mM NaCl, and 50 mM NaF) supplemented with protease inhibitors (complete; Roche) on ice. Western blots were probed with the following antibodies: to pRb, 1:1,000 (41302; BD Biosciences); to p107, 1:500 (c-18; Santa Cruz Biotechnology, Inc.); to p130, 1:2,000 (ab6545-100; Ab-Cam); to Pax7, 1:10 (Developmental Studies Hybridoma Bank [DSHB]); to myogenin, 1:10 (F5D; DSHB); to MyoD, 1:1,000 (c-20; Santa Cruz Biotechnology, Inc.); to MHC, 1:10 (MF-20; DSHB); to cyclin E, 1:1,000 (06-459; Upstate Biotechnology); to cdk2, 1:1,000 (M2; Santa Cruz Biotechnology, Inc.); and to -tubulin, 1:3,500 (T 9026; Sigma-Aldrich). Secondary detection was performed with HRP-conjugated antibodies (Bio-Rad Laboratories). Membrane-bound immune complexes were visualized by ECL Plus kit (Amersham Biosciences).
Immunostaining
Tissue samples were fixed in 4% PFA at 4°C overnight. Samples were washed in PBS for 30 min and processed for paraffin embedding. Sections were dewaxed in two washes of Citri-Solv for 5 min each and rehydrated in graded alcohol washes. Antigen retrieval was performed by immersing sections in boiling citrate buffer (0.21% wt/vol citric acid monohydrate in dH2O) for 10 min with continuous agitation. Slides were allowed to cool down to room temperature and transferred into PBS. Sections were stained with antibodies to desmin (1:200, clone D33; DakoCytomation) and to MHC (1:10, MF-20) using the fluorescent MOM kit (Vector Laboratories).
Primary myoblasts were grown on collagen-coated 4-well poly-L-lysine chamber slides (Lab-Tek). Cells were fixed with 2% PFA and permeabilized with 0.2% Triton X-100. Cells were stained with the following antibodies: to MyoD, 1:200 (M-318; Santa Cruz Biotechnology, Inc.); to MHC, 1:10 (MF-20); to myogenin, 1:10 (F5D); and to pRb, 1:250 (41302). Secondary detection was performed with fluorescein- or rhodamine-conjugated antibodies (CHEMICON International, Inc.). Cells were counterstained with DAPI and mounted in aqueous fluorescent mounting media (DakoCytomation). Bright-field, phase, and fluorescent images were viewed using an Axioplan2 fluorescent microscope (Carl Zeiss MicroImaging, Inc.) and digitally acquired (Axiocam or Axiovision 3.1; Carl Zeiss MicroImaging, Inc.) at room temperature. Image-processing software (Photoshop 7; Adobe) was used to overlay images and enhance color and clarity. Images were viewed through air-objective lenses (40x/0.75; 20x/0.5; 5x/0.12 [Carl Zeiss MicroImaging, Inc.]).
DNA synthesis assay
Primary myoblasts were grown on collagen-coated 4-well poly-L-lysine chamber slides. Cells were induced to differentiate in DM for 4 d. Before fixation, cells were pulsed with 30 µM BrdU for 1 h or restimulated in growth media supplemented with 30 µM BrdU for 24 h. Fixed cells were processed for BrdU staining using the BrdU in situ staining kit (BD Biosciences) according to the manufacturer's instructions. The final HRP-streptavidin incubation was omitted and replaced with fluorescein-conjugated streptavidin (1:200; Vector Laboratories). Cells were washed twice in PBS and double stained using rabbit antimyosin skeletal muscle (1:500, M7523; Sigma-Aldrich). Antirabbit rhodamine-conjugated secondary antibody was used to detect the myosin antibody.
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Acknowledgments |
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M.A. Rudnicki holds the Canada Research Chair in Molecular Genetics and is a Howard Hughes Medical Institute International Scholar. This work was supported by grants to M.A. Rudnicki from the Canadian Institutes of Health Research (36538), the National Institutes for Health (R01AR44031), and the Canada Research Chair Program.
Submitted: 1 March 2004
Accepted: 27 July 2004
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