Figure 3. Conditional knockout of RelA in non-ciliated epithelial Clara cells.

(A). Strategy for generating conditional knockout of RelA in non-ciliated epithelial Clara cells. RelA fl/fl conditional knockout (CKO) were crossed with an epithelial-specific Secretoglobin Family 1A Member 1 (Scgb1a1) promoter-driven inducible Cre recombinase-estrogen receptor fusion protein [CreER(™)] transgenic mouse in the C57BL6/J background. Scgb1a1-CreER(™) × RelA fl/fl mice were injected (i.p.) with oil or Tamoxifen (TMX) 10 d for epithelial RelA knockout. (B).The Scgb1a1-CreER™ × RelA^fl/fl mice crossed with the dimeric tomato (mT)/membrane EGFP (mG) mouse and exposed to vehicle oil or TMX for 10 d; 2 weeks later the lungs were analysed by IFCM. Note the membrane localized EGFP (green signal) was observed in the TMX-treated mice. (C). Validation of epithelial RelA conditional knockout. Co-staining of RelA (in green color) and epithelium-specific marker Scgb1a (in red color) were performed using sections of paraffin-embedded lung of mice were treated with oil (control) or TMX for 10 d. (D) Quantifications of total fluorescence intensities Scgb1a1 (CC10) and RelA staining. * p<0.01, n = 5. (E) Total cells and neutrophils count in the bronchoalveolar lavage fluid (BALF), expressed as total number of cells X 10³/ml (left) and percentage of neutrophils (right). #, p<0.01 compared with Oil-treated mice (n=5). (F). BALF cytokine secretion was measured using multiplex ELISA (n=5 mice per group). Protein concentration (in pg/ml) is shown. *, p<0.01, n=5.