Intrinsic Role of CFTR in Skeletal Muscle Figure 3. Abnormal calcium responses in CFTR–deficient muscle cells. (A) Negative immunostaining for CFTR in Cftr2/2 myotubes. (B) Representative Ca2+ responses in Cftr+/+ myotubes, either without (control) or with (CFTR-inh) pre-treatment by CFTR-inh172. (C) Representative Ca2+ responses in Cftr+/+ versus Cftr2/2 myotubes. (D) Lack of effect of CFTR-inh172 on the area under the curve or peak amplitude of Ca2+ responses in Cftr2/2 myotubes. doi:10.1371/journal.pgen.1000586.g003 [7,9–11]. Therefore, we compared MIP-2 (murine analogue of human IL-8) expression in Cftr+/+ and Cftr2/2 muscle cells. There were no detectable differences between wild-type and Cftr2/2 myotubes under basal conditions. However, after stimulation with pro-inflammatory cytokines (TNF-a, IL-1a, IFN-c) and Pseudomo- nas LPS, MIP-2 showed greater upregulation in Cftr2/2 diaphragmatic myotubes. This increased responsiveness also involved other pro-inflammatory chemokines such as MIP-1a and RANTES, and was similarly observed in limb muscle-derived myotubes (Figure 5A). Because many pro-inflammatory genes are regulated by NF-kB, and since the latter has been implicated in the hyperinflammatory phenotype of CF epithelial cells, we examined NF-kB (p65) binding to its DNA consensus site in nuclear extracts obtained from Cftr+/+ and Cftr2/2 diaphragmatic myotubes. As PLoS Genetics | www.plosgenetics.org 5 July 2009 | Volume 5 | Issue 7 | e1000586