Research Paper Volume 8, Issue 11 pp 2871—2896

Figure 1. Satellite cell-specific increase of NF-κB activity impairs regenerative function in vivo, but does not affect satellite cell function in vitro. (A) Biallelic SC-IKK mice contain a satellite cell specific Pax7-CreER allele and a Cre-inducible loxP-flanked STOP sequence with downstream bicistronic IKKβca-IRES-eGFP allele under control of the Rosa26 promoter. Exposure of SC-IKK mice to tamoxifen for 5 consecutive days induces Cre activity in satellite cells and causes satellite cell-specific expression of constitutively active IKKβ, leading to a satellite cell-specific increase in NF-κB activity. All mice received sodium salicylate feed or control feed starting at 2 months of age. Two weeks later, mice were injected with vehicle (corn oil) or tamoxifen, and underwent cryoinjury after an additional 4 weeks. (B) Quantification of mRNA levels of NF-κB target gene expression by qRT-PCR in satellite cells isolated from young WT mice receiving control (n=6 mice) or sodium salicylate feed (n=6 mice), or SC-IKK mice receiving control (n=5 mice) or sodium salicylate feed (n=5 mice). P-values calculated by one-way ANOVA. (C, D) Quantification of regenerating (centrally-nucleated) myofiber size in TA muscles 7 days after cryoinjury for WT mice receiving control feed (n=7 mice) or SC-IKK mice receiving control (n=7 mice) or sodium salicylate feed (n=6 mice). Mice with sodium salicylate treatment continued receiving salicylate feed during recovery after injury. Data presented as histograms of fiber size (binned by 100 μm² increments, C or as average fiber cross-sectional area (mean ± s.e.m., D). P-values determined by Kruskal-Wallis test with step-down Bonferroni method for (C) and (D). (E) Representative H&E staining of TA muscle sections at 7 days after cryoinjury from tamoxifen-treated SC-IKK and age-matched WT, with or without salicylate feed. Scale bars, 100 μm. (F) Flow cytometric analysis of satellite cell frequency in uninjured WT or SC-IKK mice receiving control or salicylate feed (n=6-9 mice per group). (G) Frequency of sorted satellite cells from uninjured WT or SC-IKK mice giving rise to myogenic colonies in clonal cell culture (n=5 or 6 mice per group). P-values were calculated by one-way ANOVA and are non-significant for all comparisons in (F) and (G). (H) Number of cells at day 8 of culture started with 1000 satellite cells isolated from tamoxifen-treated SC-IKK or WT mice (n=5 mice per group, 3 technical replicates per mouse). (I) Myogenic colony forming efficiency of satellite cells isolated from muscles of tamoxifen-treated SC-IKK or WT mice 5 days after cardiotoxin injury (n=5 mice per group). P-values calculated by Student’s t test. Data represent mean ± s.e.m., if not noted otherwise.