Fig 1.
Effect of cytokinins on differentiation of C2C12 cells.
Representative pictograms of C2C12 myoblasts grown in GM growth medium supplemented with 15% FCS for 7 days, in the presence of A) DMSO, B) 10μM trans-Zeatin, C) 1μM Kinetin, D) 10μM Kinetin, E) 1μM N6-benzyladenine, F) 10μM N6-benzyladenine. G) Fusion index was significantly higher when C2C12 cells were grown in GM in the presence of Kinetin, at 1μM and 10μM concentrations, in comparison to cells grown in the presence of GM and DMSO. Similarly, 1μM and 10μM BA increased the Fusion index in comparison to cells grown in the presence of GM and DMSO. As a positive control, C2C12 cells were grown in DM supplemented with 2%HS for 4 days. H) Kinetin at 1μM and 10μM concentration, and N6-benzyladenine at 1μM and 10μM concentration, significantly induced MCK activity in comparison to cells grown in DM and DMSO. Error bars are ± SEM (n = 9). One-way Anova with Bonferroni post hoc test: *p<0.05, **p<0.01, ***p<0.001. Black arrows indicate apparent presence of myotubes. Scale bar 50μm. FCS (Foetal Calf Serum); HS (Horse Serum); DM (Differentiation medium); GM (Growth medium); K (Kinetin); BA (N6-benzyladenine); ZEA (trans-Zeatin); MCK (Muscle Creatine Kinase).
Fig 2.
Kinetin but not N6-benzyladenine (BA) increases C2C12 differentiation in the DM differentiation medium.
C2C12 myoblasts were grown in DM supplemented with 2% horse serum in the presence of DMSO, 1μM or 10μM Kinetin, 1μM or 10μM BA. Kinetin but not BA increases A) Myog mRNA levels and B) MCK activity, 3 days post induction of myoblast differentiation. This effect was diminished 6 days post induction of myoblast differentiation, when C) Myog mRNA levels and D) MCK activity were not elevated in the presence of either Kinetin or N6-benzyladenine. Error bars are ± SEM (n = 6). One-way Anova with Bonferroni post hoc test: *p<0.05, **p<0.01, ***p<0.001. HS (Horse Serum); DM (Differentiation Medium); K (Kinetin); BA (N6-benzyladenine); MCK (Muscle Creatine Kinase); Myog (Myogenin).
Fig 3.
Effect of Kinetin on C2C12 myoblast differentiation in the mitogen enriched medium.
A) Fusion index of C2C12 cells increased significantly in the presence of Kinetin at the 0.1μM, 1μM, 10μM and 100μM concentrations, in comparison to cells grown in the presence of GM and DMSO. B) MCK activity and C) Myog transcript levels were significantly higher in the presence of Kinetin at the 0.1μM, 1μM, 10μM and 100μM concentrations, in comparison to cells grown in the presence of GM and DMSO. Error bars are ± SEM (n = 6). One-way Anova with Bonferroni post hoc test: *p<0.05, **p<0.01, ***p<0.001. Scale bar 100 μm. FCS (Foetal Calf Serum); DM (Differentiation medium); GM (Growth medium); K (Kinetin); BA (N6-benzyladenine); MCK (Muscle Creatine Kinase); Myog (Myogenin).
Fig 4.
Kinetin riboside but not Kinetin is toxic in C2C12 cells.
Kinetin but not Kinetin riboside supplement increased significantly A) fusion index and B) MCK activity, in C2C12 cells grown in GM with 15% FCS, in comparison to C2C12 myoblasts supplemented with DMSO only for 7 days. C) An MTT assay showed that Kinetin riboside but not Kinetin was toxic in C2C12 cells, at the tested concentrations, after 3 days in culture. Error bars are ± SEM (n = 6). One-way Anova with Bonferroni post hoc test: *p<0.05, **p<0.01, ***p<0.001. FCS (Foetal Calf Serum); DM (Differentiation medium); K (Kinetin); KR (Kinetin riboside); GM (Growth Medium); MCK (Muscle Creatine Kinase).
Fig 5.
Time-dependent effect of Kinetin on C2C12 differentiation into myotubes.
C2C12 cells were grown in GM supplemented with DMSO (negative control) or with different concentrations of Kinetin at 0.1μM, 1μM and 10μM. Myog transcript levels and MCK activity was validated at 3 days (A and B), 5 days (C and D) and 7 days (E and F) post seeding C2C12 myoblasts. Error bars are ± SEM (n = 6). One-way Anova with Bonferroni post hoc test: *p<0.05, **p<0.01, ***p<0.001. FCS (Foetal Calf Serum); HS (Horse Serum); DM (Differentiation medium); GM (Growth medium);K (Kinetin); MCK (Muscle Creatine Kinase). Myog (Myogenin).
Fig 6.
Kinetin enhances MYOD-dependent conversion of 10T1/2 fibroblasts into myotubes.
10T1/2 fibroblasts were transiently transfected with a full-length MyoD gene expression construct and were grown in DM supplemented with 5% horse serum. 24 hours post transfection, cells were supplemented with either DMSO (negative control) or Kinetin at 0.1μM, 1μM or 10μM concentration, and were grown for 6 days. Kinetin supplemented at the 1μM and 10μM concentrations significantly increased A) Myog transcript levels and B) MCK activity, which correlates with an increased conversion of fibroblasts into myotubes. Error bars are ± SEM (n = 8). One-way Anova with Bonferroni post hoc test: *p<0.05, **p<0.01, ***p<0.001. K (Kinetin); MCK (Muscle Creatine Kinase); Myog (Myogenin); DM–Differentiation Medium.