Figure 3: The TLR5/NAIP5 pathway of NLRC4 activation is defective in murine CF.

RAW 247.3 macrophages were exposed to (a) A. fumigatus live conidia before the assessment for NLRP3 or NLRC4 protein expression at different times by immunoblotting with specific antibodies or to (b) various fungal antigens for 2 h at 37 °C before NLRC4 protein expression by western blotting. (c) Cells were pretreated with specific siRNA for Tlr5, Naip2, Naip5 or scrambled siRNA (siScram) or the NF-κB inhibitor SN50 (100 μM), exposed to Aspergillus conidia or flagellin for 2 h at 37 °C and assessed for NLRC4 protein expression by western blotting. All immunoblots were normalized against the corresponding β-actin. (d) C57BL/6 mice were given siRNA intranasally twice, 2 days before and 3 days after the infection before the assessment of NLRC4 protein expression at 4 dpi. (e) Tlr5, Naip2 and Naip5 expression by RT–PCR in lungs of C57BL/6 and Cftr^−/− mice infected with A. fumigatus or P. aeruginosa (n=6 for all groups) 3 days before. (f) NLRC4 and phospho(p)NLRC4 expression in purified lung marcrophages from C57BL/6 and Cftr^−/− mice stimulated with live A. fumigatus conida or P. aeruginosa at the ratio cells:microbes (1:1) in the presence of EDTA at 37 °C. The expression of pNLRC4 was normalized against the corresponding β-actin. Data pooled from three experiments and presented as mean±s.d. for all bar graphs. *P<0.05, ***P<0.001, ****P<0.0001, None versus infected mice, Two-way ANOVA, Bonferroni post hoc test.