Article Figures & Data
Figures
- Fig. 1.
IGF-I and PDGF maintain myoblast survival, but only IGF-I promotes differentiation. (A) Cell counts (expressed as the percentage at time zero [T0]) of C2AS12 myoblasts after a 24- or 48-h incubation in DM with or without R3IGF-I (2 nM) or PDGF-BB (0.4 nM). The asterisk indicates that significantly fewer cells survived (P < 0.0006) in untreated than in growth factor-treated cells. Results are means ± SEMs from three experiments, each performed in duplicate. (B) IGF-I induces muscle differentiation. Results of immunocytochemical staining for myogenin and MHC after incubation of C2AS12 myoblasts in DM with PDGF-BB or R3IGF-I for 24 to 72 h are shown.
- Fig. 2.
Treatment with IGF-I stimulates expression of p21. (A) The autoradiograph shows results of a representative RNase protection assay performed using total RNA isolated from C2AS12 cells incubated with either R3IGF-I (2 nM) or PDGF-BB (0.4 nM) for the indicated times. The numbers below each panel correspond to the change in mRNA abundance relative to that at time zero. The bottom panel shows a photograph of an ethidium bromide-stained gel of the RNA used in these studies. Similar results were seen in three independent experiments. (B) The upper panel shows a representative immunoblot using an antibody to p21 and whole-cell protein extracts from C2AS12 cells incubated with either R3IGF-I (2 nM) or PDGF-BB (0.4 nM) for the indicated times. The numbers below each panel correspond to the change in protein abundance relative to that at time zero. Similar results were seen in seven independent experiments. The lower panel shows the same blot after being stripped and incubated with an antibody to CDK-4. (C) Representative fluorescence micrographs of C2AS12 myoblasts treated with either R3IGF-I (2 nM) or PDGF (0.4 nM) for 24 h as described in Materials and Methods and stained with anti-p21 antibody (A and B) or Hoechst dye (C and D). Panels E and F are merged images of panels A and C and panels B and D, respectively.
- Fig. 3.
Forced expression of p21 stimulates myoblast survival. C2AS12 cells were transfected with the p21-IRES-EGFP expression plasmid shown above the graph, or with EGFP alone, as described in Materials and Methods. Cell counts of transfected myoblasts were performed after a 24- or 48-h incubation in DM or in DM supplemented with R3IGF-I (2 nM). Results are presented as means ± SEMs from four experiments, each performed in duplicate. The asterisks indicate that survival was significantly less in myoblasts transfected with EGFP than in p21-transfected or IGF-I-treated cells (∗, P < 0.001, ∗∗, P < 0.003). CMV/EP, enhancer-promoter from cytomegalovirus.
- Fig. 4.
Inhibition of p21 expression prevents IGF-stimulated myoblast survival. (A) Expression of a p21AS cDNA blocks IGF-induced accumulation of p21 protein. C2AS12 cells were transiently cotransfected with the p21AS-IRES-EGFP and EGFP expression plasmids, as described in Materials and Methods. Cells were then incubated in DM plus R3IGF-I (2 nM) for 24 h. The top panels show photomicrographs of a group of cells (magnification, ×400). The left panel shows expression of EGFP, and the center panel shows results of immunostaining for p21. In the right panel merged EGFP and p21 images are displayed. Below the micrographs results are presented as the percentage of cells transfected with either the p21AS-IRES-EGFP or IRES-EGFP expression plasmid that also express p21 protein (means ± SEMs from three experiments, counting 100 cells per experiment; ∗, P < 0.0001). (B) Inhibition of p21 blocks IGF-mediated myoblast survival. C2AS12 cells were transiently transfected with the p21AS-IRES-EGFP expression plasmid. Cell counts of transfected myoblasts were performed at 24 and 48 h after incubation in DM or in DM supplemented with R3IGF-I (2 nM) or PDGF-BB (0.4 nM). Results are presented as the means ± SEMs from three independent experiments, each performed in duplicate. The symbols indicate that survival was significantly less in myoblasts transfected with p21AS and treated with DM or R3IGF-I than in cells transfected with p21AS and incubated with PDGF-BB at 24 h (∗, P < 0.01; ∗∗, P < 0.03) or at 48 h (#, P < 0.02; ##, P < 0.001).
- Fig. 5.
Akt induces p21 expression. (Top panels) Representative fluorescence micrographs of C2AS12 muscle cells transiently transfected with the iAkt-IRES-EGFP expression plasmid, as described in Materials and Methods. Following a 24-h incubation in DM alone (A and C) or in DM containing HT (1 μM) (B and D), expression of iAkt was assessed by immunostaining using an anti-HA (α-HA) primary antibody and a fluorescein-labeled secondary antibody (A and B). Expression of p21 (C and D) also was determined by immunofluorescence. (Bottom panel) The graph shows quantitation of p21 expression in cells transfected with the iAkt-IRES-EGFP plasmid following treatment with DM alone or containing HT (means ± SEMs from three experiments, counting 100 cells per experiment; ∗, P < 0.003).
- Fig. 6.
IGF-I treatment does not cause nuclear translocation of the NF-κB subunit p65 Rel A. (A to C) Representative fluorescence micrographs of C2AS12 myoblasts incubated in DM without or with either R3IGF-I (2 nM) or tumor necrosis factor alpha (1.2 nM) for 2 h and immunostained with an antibody to p65 Rel A. (D to F) Same cells as in panels A to C stained with Hoechst dye.
- Fig. 7.
Inhibition of p21 expression blocks Akt-mediated myoblast survival. (A) C2AS12 cells were transiently cotransfected with iAkt-IRES-EGFP and either the p21AS-IRES-EGFP or EGFP expression plasmid, as described in Materials and Methods. Cell counts of transfected myoblasts were performed 24 h after incubation in DM or in DM plus HT (1 μM). Results are presented as the means ± SEMs from three independent experiments, each performed in duplicate. Survival was significantly greater in HT-treated cells cotransfected with EGFP than with p21AS (∗, P < 0.003). (B) Inhibition of p21 expression does not block induction of Akt kinase activity. Results are of in vitro kinase assays for Akt using immunoprecipitates from cells cotransfected with the iAkt-IRES-EGFP and p21AS-IRES-EGFP expression plasmids and incubated without or with HT for 4 h are shown. Results from a representative experiment are pictured in the top panel. Results of three independent experiments (means ± SEMs) are plotted in the bottom panel. Values on the y axis represent arbitrary densitometric units. Significantly more Akt enzymatic activity could be measured after treatment with HT (∗, P < 0.003).
- Fig. 8.
IGF-I cannot maintain survival in the absence of p21 expression. C2AS12 cells were transiently cotransfected with p21AS-IRES-EGFP and iAkt-IRES-EGFP expression plasmids, as described in Materials and Methods. Cell counts of transfected myoblasts were performed 24 h after incubation in DM or in DM supplemented with R3IGF-I (2 nM) or PDGF-BB (0.4 nM). Results are presented as the means ± SEMs from three independent experiments, each performed in duplicate. The asterisks indicate that survival was significantly less in transfected myoblasts treated with IGF-I than in cells incubated with PDGF (P < 0.01).
- Fig. 9.
Inhibition of p21 expression blocks muscle cell survival. C2 or L6 myoblasts were transiently transfected with the p21AS-IRES-EGFP or EGFP expression plasmid. Cell counts of transfected myoblasts were performed at 24 and 48 h after incubation in DM. Results are presented as the means ± SEMs from three independent experiments, each performed in duplicate. The asterisks indicate that survival was significantly less in myoblasts transfected with p21AS-IRES-EGFP than in cells transfected with EGFP at either time point (∗, P < 0.01; ∗∗, P < 0.03).
