FIGURE 5.

IL-6 deficiency impairs macrophage-stimulated myoblast proliferation. A, BrdU immunohistochemical staining was used to detect the proliferating cells in injured muscle (at day 5 after injury) of WT and IL-6^−/− mice, the right graph indicated the percentages of positive areas (n = 3 in each group; scale bars, 50 μm). B, double immunofluorescence staining with BrdU (green) and MyoD (red) in injured muscle (at day 5 after injury) of WT mice, nuclei were stained with DAPI (blue) (scale bars, 50 μm). C, cyclin D1 mRNA levels of injured muscle (5 days after injury) of WT and IL-6^−/− muscle were valued by qRT-PCR (n = 3 in each group). *, p < 0.05, compared with WT values. D, rIL-6 (0, 0.1, 1, or 10 ng/ml) and IL-6 neutralization antibody (10 μg/ml) were added to the medium of C2C12 myoblasts, and proliferation was measured by BrdU-positive cells. E, during 48 h of co-culture, C2C12 myoblasts (1 × 10⁴/well) and WT BMDMs (1 × 10³, 2 × 10³, 1 × 10⁴, 5 × 10⁴) were separated by the Transwell membrane, and the proliferation states of myoblast were detected by BrdU incorporation assay. *, p < 0.05; **, p < 0.01, compared with PBS control group. F, the proliferation of C2C12 myoblasts co-cultured with WT or IL-6^−/− BMDMs (5 × 10⁴/well) were measured by the BrdU incorporation ratio. The left graph was the representation of cultured cells, and the right graph was the statistical result. G, the cytokines in the culture medium of WT and IL-6^−/− BMDMs were analyzed by Bio-plex. *, p < 0.05, compared with WT values. Data were the presentation of three independent experiments. CTX, cardiotoxin.