Interleukin-6 Induces Myogenic Differentiation via JAK2-STAT3 Signaling in Mouse C2C12 Myoblast Cell Line and Primary Human Myoblasts

Figure 1. Effect of low, medium and high IL-6 concentrations on C2C12 cell cycle progression at 24 h. (A) Flow cytometric analysis of IL-6-treated cells to determine the effect of IL-6 concentration on C2C12 cell cycling was done after 24 h incubation (B) C2C12 cells (%) in each cell cycle stage after treatment of cells with low, medium and high IL-concentrations for 24 h. The experiment was repeated three times. Results are presented as mean ± standard error of the mean (S.E.M). Statistical analysis: Analysis of variance (ANOVA) and Bonferroni post hoc test. * p < 0.05; relative to control.

Figure 2. Effect of low, medium and high IL-6 concentrations on PCNA, MyoD and myogenin protein levels in C2C12 cells. (A) PCNA protein levels in C2C12 cells treated with low, medium and high IL-6 concentrations for 24 h. (B) MyoD protein levels in C2C12 cells treated with low, medium and high IL-6 concentrations for 24 h. (C) Immunofluorescence staining for PCNA and myogenin in C2C12 cells treated with low, medium and high IL6 concentrations for 24 h. The experiment was repeated three times. Immunofluorescence images were taken at 100× magnification. Results are presented as mean ± standard error of the mean (S.E.M). Statistical analysis: ANOVA and Bonferroni post hoc test. * p < 0.05.

Figure 3. Effect of low, medium and high IL-6 concentration on C2C12 cell cycling after 48 h of treatment. (A) Flow cytometric analysis of IL-6-treated cells to determine the effect of low, medium and high IL-6 concentrations on C2C12 cell cycling was done after 48 h incubation (B) C2C12 cells (%) in each cell cycle stage after treatment of cells with low, medium and high IL-6 concentrations for 48 h. The experiment was repeated three times. Results are presented as mean ± standard error of the mean (S.E.M). Statistical analysis: ANOVA and Bonferroni post hoc test. * p < 0.05; relative to control.

Figure 4. Concentration-dependent effect of IL-6 on C2C12 myogenic regulatory factors’ protein levels. (A) Evaluation of PCNA, MyoD and myogenin protein levels in C2C12 cells treated with low, medium and high IL-6 concentrations for 48 h. (B) Quantification of PCNA protein levels as shown in (A). (C) Quantification of MyoD protein levels as shown in (A). (D) Quantification of myogenin protein levels as shown in (A). (E) Immunofluorescence staining for PCNA and myogenin in C2C12 cells after treatment with low, medium and high IL-6 concentrations for 48 h. The experiment was repeated three times. Immunofluorescence images were taken at 100× magnification. Results are presented as mean ± standard error of the mean (S.E.M). Statistical analysis: ANOVA and Bonferroni post hoc test. * p < 0.05.

Figure 5. IL-6 activates the STAT3 signaling pathway in C2C12 cells. (A) Evaluation of p-STAT3 protein levels in C2C12 cells at 5 min up to 30 min after treatment of cells with low, medium and high IL-6 concentrations. (B) Quantification of western blot images shown in (A). (C) Effect of JAK-STAT inhibitor, AG490, on p-STAT3 expression in C2C12 cells over 15 min after treatment with low and high IL-6 concentration. (D) Effect of JAK-STAT inhibitor, AG490, on PCNA, MyoD and myogenin protein levels in C2C12 cells after treatment with low and high IL-6 concentrations (top panel) and densitometric quantification of images (lower panel) (E) Quantification of PCNA, MyoD and myogenin protein levels in C2C12 cells after treatment with low and high IL-6 concentrations plus JAK-STAT inhibitor, AG490. The experiment was repeated three times. Immunofluorescence images were taken at 100× magnification. Results are presented as mean ± standard error of the mean (S.E.M). Statistical analysis: ANOVA and Bonferroni post hoc test. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 6. Effect of IL-6 concentration and JAK-STAT inhibitor on C2C12 cell cycling at 48 h. (A) Flow cytometric analysis of IL6-treated cells, with or without JAK-STAT inhibitor, to determine the effect of IL-6 concentration on C2C12 cell cycling was done after 48 h of incubation. Displayed are representative images (B) Summary of data shown in (A) of C2C12 cells after treatment with IL-6 and JAK-STAT inhibitor, AG490. The experiment was repeated three times. Results are presented as mean ± standard error of the mean (S.E.M). Statistical analysis: ANOVA and Bonferroni post hoc test.

Figure 7. The phenotypic characterization of primary human myoblasts. (A–D) Flow cytometric analysis of Primary Human myoblasts (PHMs) was performed as described in Materials and Methods section. Average of cells (%) staining positive for Pax7 and desmin are shown. (E) Several preparations of the PHMs were analyzed and shown is representative of eight donors. Results are shown as mean ± S.D. (F) Red outlined column (first column): Preliminary characterization of isolated cells as performed to ascertain whether the culture generated contained myoblasts: Pax7 (green), desmin (red) (fluorescent microscopy at 100× magnification). Green outlined Column (second column): PHMs were exposed to differentiation inducing conditions for 7 days and stained via immunofluorescence for desmin (red), myogenin (green) and nuclei (blue). White arrows indicate multinucleated myofibres. Blue outlined Column (third column): PHMs were able to preserve their differentiation ability after long periods of sub-culturing. Cells were maintained and passaged for two months (imaged by confocal microscopy (myogenin) at 400× magnification or fluorescent microscopy (Trans) at 200× magnification). (G) Comparison of C2C12 and…

Figure 8. Concentration- and time-dependent effects of low and high IL-6 concentrations on PCNA, MyoD and myogenin expression in PHMs. (A) Western blot determination of PCNA, MyoD and myogenin protein levels 24 h after treatment with low and high IL-6 concentrations. (B) Western blot determination of PCNA, MyoD and myogenin protein levels 48 h after treatment with low and high IL-6 concentrations. (C) Quantification of PCNA, MyoD and myogenin protein levels as shown in (A). (D) Quantification of PCNA, MyoD and myogenin protein levels as shown in (B). (E) Immunofluorescence staining for PCNA, MyoD and myogenin protein in PHMs after treatment with low and high IL-6 for 24 and 48 h. The experiment was repeated three times. Immunofluorescence images were taken at 100× magnification. Results are presented as mean ± standard error of the mean (S.E.M). Statistical analysis: ANOVA and Bonferroni post hoc test. * p < 0.05; *** p < 0.001.

Figure 9. Effect of low and high IL-6 concentrations on JAK1 and JAK2 phosphorylation in PHMs. (A) Effect of low and high IL-6 concentration on JAK1 phosphorylation over 8 h. (B) Effect of low and high IL-6 concentration on JAK2 phosphorylation over 8 h. (C) Quantification of p-JAK1 as shown in (A). (D) Quantification of p-JAK2 as shown in (B). (E) Immunofluorescence staining for p-JAK1 after treatment of PHMs with low and high IL-6 concentration over 8 h. (F) Immunofluorescence staining for p-JAK2 after treatment of PHMs with low and high IL-6 concentration over 8 h. (G) Immunofluorescence staining for p-STAT3 after treatment of PHMs with low and high IL-6 concentration over 8 h (H) Quantification of p-STAT3 levels as shown in (G). The experiment was repeated three times. All immunofluorescence images were taken at 100× magnification. Results are presented as mean ± standard error of the mean (S.E.M). Statistical analysis: ANOVA and Bonferroni post hoc test. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 10. Concentration- and time-dependent effects of low and high IL-6 concentrations on SOCS1 and SOCS3 protein levels in PHMs. (A) Western blot determination of SOCS3 and SOCS1 protein levels 24 h after treatment with low and high IL-6 concentrations. (B) Western blot determination of SOCS3 and SOCS1 protein levels 48 h after treatment with low and high IL-6 concentrations. (C) Quantification of SOCS3 and SOCS1 protein levels as shown in (A). (D) Quantification of SOCS3 and SOCS1 protein levels as shown in (B). (E) Immunofluorescence staining for SOCS3 and SOCS1 after treatment of PHMs with low and high IL-6 concentration for 48 h. The experiment was repeated three times. Immunofluorescence images were taken at 100× magnification. Results are presented as mean ± standard error of the mean (S.E.M). Statistical analysis: ANOVA and Bonferroni post hoc test. * p < 0.05; ** p < 0.01.

Figure 11. Concentration- and time-dependent effects of low and high IL-6 concentrations on IL-6R expression in PHMs. (A) Western blot determination of IL-6R protein levels 24 h after treatment with low and high IL-6 concentrations. (B) Western blot determination of IL-6R protein levels 48 h after treatment with low and high IL-6 concentrations. (C) Quantification of IL-6R protein levels as shown in (A). (D) Quantification of IL-6R mRNA levels after treatment of cells with low and high IL-6. (E) Real time PCR analysis of IL-6R mRNA transcripts 24 h after treatment of PHMs with low and high IL-6 concentration. (E) Real time PCR analysis of IL-6R mRNA transcripts 48 h after treatment of PHMs with low and high IL-6 concentration. (E) Immunofluorescence staining for IL-6R after treatment of PHMs with low and high IL-6 concentration for 24 (top panel) and 48 h (lower panel). The experiment was repeated three times. Immunofluorescence images were taken at 100× magnification. Results are presented as mean ± standard error of the mean (S.E.M). Statistical analysis: ANOVA and Bonferroni post hoc test. * p < 0.05; ** p < 0.01.

Figure 12. Schematic summary of IL-6 induced effects on the JAK-STAT pathway and its main regulator, SOCS. Low IL-6 concentration depicted on the left side and high IL-6 concentration on the right. Green arrows indicate findings of this study.