Irisin promotes C2C12 myoblast proliferation via ERK-dependent CCL7 upregulation
Fig 4
CCL7 promotes C2C12 cell proliferation.
(A) Treatment with murine CCL7 but not C3 recombinant protein promoted C2C12 cell proliferation. The cells were serum-straved for 4 hours and then treated with C3, CCL7, or irisin for 24 hours. Cell proliferation (%) was measured by MTS assay. Data are represented as the means ± SD (n = 3). Asterisk symbols (*, **, and ***) indicate significant differences between control group and C3, CCL7 or irisin-treated groups (p < 0.05, p < 0.01, and 0.001, respectively). (B) Knockdown of CCL7 suppresses irisin-induced C2C12 cell proliferation. Cells were transfected with scrambled (negative control) or indicated siRNA for 48 hours with or without treatment of irisin. Cell proliferation (%) was measured by MTS assay. Data are represented as the means ± SD (n = 4). An asterisk symbol (**) indicates significant difference between control group and irisin-treated group (p < 0.01) in C2C12 cells transfected with scrambed siRNA. n.s, not significant (p < 0.05). (C) Knockdown of Ccl7 diminished Ccl7 mRNA expression in C2C12 cells. Cells were transfected with scrambled (negative control) or indicated siRNA for 48 hours. Relative mRNA levels were analyzed by qRT-PCR using SYBR Green dye (n = 3 per each group). Data are represented as the means ± SD (n = 4). An asterisk symbol (***) indicates significant difference between negative control group (scrambled) and siCCL7 groups (p < 0.001). (D) Knockdown of Ccl7 suppressed gene expression of the cell proliferation markers Pnca, Mki67, and Mcm2 in C2C12 cells. The C2C12 cells were transfected with scrambled (negative control) or indicated siRNA for 48 hours, then, the cells were treated with 200 ng/mL of irisin for 24 hours. Relative mRNA levels were measured by qRT-PCR. Bar graph indicates mean ± SD (n = 3). An asterisk symbols (**) indicates significant difference between control group and irisin-treated groups (p < 0.01). n.s, not significant (p > 0.05).