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).