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First published online 16 November 2004
doi: 10.1242/jcs.01548

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Stem cell antigen-1 is necessary for cell-cycle withdrawal and myoblast differentiation in C2C12 cells

¹ Cardiovascular Research Institute, University of California, San Francisco, CA 94143, USA
² Department of Pediatrics, University of California, San Francisco, CA 94143, USA
³ Cancer Center, University of California, San Francisco, CA 94143, USA
⁴ Life Sciences Division, Genome Sciences, Ernest Orlando Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA

View larger version (29K): [in a new window]   Fig. 1. Sca-1 is expressed on a subpopulation of C2C12 cells and is removed by PIPLC. (A) Flow cytometry of proliferating C2C12 cells (Pro; green) and cells grown in differentiation media for two days (Day 2; red) demonstrated that the percentage of cells with surface expression of Sca-1 increases during differentiation, but that Sca-1-positive and -negative populations persist. Gates (vertical gray bar) were established by nonspecific PE-conjugated antibody binding in each experiment (black and gray lines). A representative profile is shown with mean values for percent Sca-1-positive and -negative cells given (n=3; *significant difference, P<0.05). (B) Immunoblot analysis of Sca-1 with specific monoclonal antibody E13-161.7 in proliferating C2C12 cells (Pro) and cells grown in differentiation media for 2 and 5 days demonstrated removal of Sca-1 protein from whole cell lysates with PIPLC (5 U/ml). ß-actin was used as a control. Representative data are shown (n=4). (C) Immunostaining of Sca-1 with monoclonal antibody E13-161.7 demonstrated stripping of Sca-1 (green) from the cell surface with PIPLC (5 U/ml). Bar, 50 µm.

View larger version (62K): [in a new window]   Fig. 2. PIPLC inhibits cell-cycle withdrawal and myotube formation in rodent and avian myoblasts. C2C12 (A-C), Sol8 (D-F), L6 (G-I), and QM7 (J-L) myoblasts were grown under differentiation conditions in the presence and absence of PIPLC (5 U/ml) until extensive myotube formation was observed in untreated cells: 5 days for C2C12 and Sol8 cells, 7 days for QM7 cells, and 10 days for L6 cells. All cultures were then labeled with BrdU. (A,D,G,J) Detection of all nuclei with DAPI and nuclei of cycling cells by immunostaining for BrdU demonstrated an increase in cycling cells and a decrease in multinucleated myotubes (as determined by phase microscopy) in the presence of PIPLC. Representative data are shown (n=5). Bar, 50 µm. (B,E,H,K) Fusion index [number of nuclei in fused cells (2 nuclei)/total nuclei] was decreased in cells treated with PIPLC, compared with untreated cells. Data shown are mean±s.e.m. (n=4; *significant difference, P<0.05). (C,F,I,L) Percent BrdU-positive cells was increased in cells treated with PIPLC, compared with untreated cells. Data shown are mean±s.e.m. (n=5; *significant difference, P<0.05).

View larger version (32K): [in a new window]   Fig. 3. Sca-1 is essential for myoblast cell-cycle withdrawal and myotube formation during differentiation. (A) Fusion index [number of nuclei in fused cells (2 nuclei)/total nuclei] was decreased in cells grown for 5 days in differentiation media in the presence of E13-161.7 and D7 antibodies, compared with untreated cells (CNT) or cells treated with either antibody alone or antibody against CD34, a primitive stem cell marker also found on C2C12 myoblasts. Data shown are mean±s.e.m. (n=3; *significant difference, P<0.05). (B) Percent BrdU-positive cells was increased in cells grown for 5 days in differentiation media in the presence of E13-161.7 and D7 antibodies, compared with untreated cells (CNT) or cells treated with either antibody alone or anti-CD34 antibody. Data shown are mean±s.e.m. (n=3; *significant difference, P<0.05). (C) Immunoblot analysis demonstrated downregulation of Sca-1 expression in C2C12 cells stably transfected with Sca-1 antisense (AS), compared with cells transfected with empty vector (SHAM). ß-actin was used as a control. Typical results from clone 6 are shown. (D) Flow cytometry demonstrated a decrease in the subpopulation of Sca-1-expressing C2C12 cells in cultures grown for 2 days in differentiation medium (Day 2) and stably transfected with Sca-1 antisense (AS; orange), compared with cells transfected with empty vector (S; red) (*significant difference, P=0.035). There was a small, but not statistically significant, decrease in the percent Sca-1-expressing cells in proliferating cultures (Pro) stably transfected with Sca-1 antisense (AS; blue), compared with cells transfected with empty vector (S; green) (P=0.4). Gates (vertical gray bar) were established by nonspecific PE-conjugated antibody binding in each experiment. A representative profile is shown with mean values for percent Sca-1-positive and -negative cells given (n=3). (E) Fusion index [number of nuclei in fused cells (2 nuclei)/total nuclei] was decreased in cells expressing Sca-1 antisense (AS) and grown for 5 days in differentiation media, compared with cells expressing empty vector (SHAM). Data shown are mean±s.e.m. (n=4; *significant difference, P<0.05) (F) Percent BrdU-positive cells was increased in cells expressing Sca-1 antisense (AS) and grown for 5 days in differentiation media, compared with cells expressing empty vector (SHAM). Data shown are mean±s.e.m. (n=4; *significant difference, P<0.05).

View larger version (61K): [in a new window]   Fig. 4. Sca-1 downregulation blocks myogenesis. Untransfected, control C2C12 cells (C) and cells expressing Sca-1 antisense (AS) were grown under proliferation conditions and in differentiation medium for 5 days (as described for Fig. 3). They were then metabolically labeled with BrdU and stained with DAPI to identify nuclei, with antibody against BrdU (red) to detect DNA synthesis, and with antibodies against Myf5 (green), MyoD (green) and myogenin (green) as markers of myoblast differentiation. Bar, 50 µm. (A) Whereas proliferating (Pro) control and antisense cells expressed Myf5 and MyoD, and myogenin was expressed under differentiation conditions in both cell lines, expression of the early myogenic marker Myf5 persisted in Sca-1 antisense cells under differentiation conditions. Quantitative analysis provided in text. (B) Wild-type cells were negative for Myf5 and BrdU after 5 days in differentiation medium, whereas Sca-1 antisense cells grown in differentiation medium for 5 days were positive for both Myf5 and BrdU. Quantitative analysis provided in text.

View larger version (19K): [in a new window]   Fig. 5. Fyn mediates Sca-1 function in myogenesis. (A) Fyn and Src kinase activities were measured in wild-type C2C12 cells (C; white bars) and cells expressing Sca-1 antisense (AS; gray bars). Cells grown in differentiation media for 2 and 5 days demonstrated an appropriate increase in Fyn activity in control and antisense-expressing cells at day 5 (top), low levels of Src activity at all time points (bottom), and an inappropriate increase in Fyn activity at day 2 in cells expressing Sca-1 antisense (top). Data shown are mean±s.e.m. (n=3; *significant difference, P<0.05). Pro, proliferating cells. (B) Control (solid white or grey) or mutant-expressing plasmids (hashed bars), both co-expressing GFP, were transfected into Sca-1 antisense (AS) or wild-type C2C12 (C) cells. Cells were grown in differentiation media for 5 days. The ratio of GFP-expressing (i.e. transfected) mononuclear cells:myotubes (2 nuclei) was lower in antisense cells (AS) transfected with dominant-negative Fyn-K299M compared with antisense cells transfected with control plasmid expressing GFP alone (GFP), and higher in wild-type (C) cells transfected with constitutively active Fyn-Y531F compared with GFP-transfected wild-type cells. Data shown are mean±s.e.m. (n=3; *significant difference, P<0.05).

View larger version (18K): [in a new window]   Fig. 6. Sca-1 is expressed on a subpopulation of primary murine skeletal myoblasts. Flow cytometry of primary myoblasts isolated from the hindlimbs of C57BL/6 mice and cultured in differentiation medium (DM) for at least 3 days demonstrated that a subpopulation of Sca-1-positive cells and small myotubes, constituting 25% of selected myoblasts at the time of isolation, decreased in number with differentiation and expression of myosin (top). Of particular note, Sca-1 expression in this residual population of Sca-1 expressors increased on a per cell basis (middle), whereas the size of these cells, as a function of forward angle light scatter (FALS), remained constant (bottom). All data points are shown. Trendlines were generated by bivariate regression analysis using the Pearson coefficient with significance levels indicated.

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