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. 2015 Jun 3;195(2):683–694. doi: 10.4049/jimmunol.1402983

FIGURE 2.

FIGURE 2.

CARMA3 mediates proinflammatory cytokine release from normal human bronchial epithelial cells in response to GPCR stimulation. (A) A lentivirus containing a shRNA against CARMA3 and a puromycin resistance cassette was used to infect NHBE cells (multiplicity of infection of 0.25). The infected cells were selected for puromycin resistance for 5 d and then Western immunoblotting was used to detect CARMA3. (B–F) NHBEs were grown on an ALI postinfection with lentivirus containing a nontargeting shRNA (scRNA) or a CARMA3-targeting shRNA. These cells were then stimulated with either 10 μM LPA, 100 μg/ml Alternaria, 100 μM ATP, or 100 μg/ml HDM for 6 h. RNA was then isolated from the cells and the levels of (B) CARMA3, (C) CXCL8/IL-8, (D) GM-CSF, (E) CCL20/MIP-3α, and (F) TSLP were determined. GAPDH levels were used to normalize the values of genes tested. Data are expressed as fold change from media. Values are the mean of three to six samples ± SEM. This experiment was repeated twice. *p < 0.05 versus scRNA cells by unpaired t test. ND, not detected.