(A) Confocal image (left) shows overlay of the indicated fluorescence labels on sessile AMs. Dotted lines indicate alveoli in which sessile AMs are located. Selected AMs (rectangle), imaged at high magnification (middle and right images), show TNF-α fluorescence before and after hyperinflation. The tracings depict fluorescence quantification at the indicated times. Findings were replicated in at least 3 AMs each in 3 lungs. (B) Confocal image (left) shows overlay of the indicated fluorescence labels on sessile AMs from same imaging field as A. Dotted lines indicate alveolar epithelial margins. Confocal images (middle and right images), show calcein fluorescence alone after laser bleach at indicated times. Dashed circles indicate laser bleached regions. Regression plot shows data for individual AMs from 3 lungs. Same color indicates AMs from an individual lung. TNF-α secretion is quantified as percentage fluorescence decrease from baseline. Line drawn by linear regression analysis. GJC, gap junction communication; FRAP %, fluorescence recovery after photobleaching expressed as difference from initial fluorescence, quantified 60 minutes after bleaching. (C) Confocal images of epithelial and TNFR1 fluorescence in alveoli microinfused with calcein (green) and anti-TNFR1 mAb (red) in littermate control mice (Cx43^FL) and in mice with AM-specific Cx43 knockout (AM-Cx43^KO) as indicated. Bars show TNFR1 shedding response. (D) Confocal image (left) shows overlay of the indicated fluorescence labels on sessile AMs. Selected AM (purple rectangle in the left image), imaged at high magnification, shows TNF-α fluorescence before and after hyperinflation in the absence (Buffer) or presence of XeC. Bars show quantification of TNF-α secretion. Data analyses. Group data are mean ± SEM. P values were calculated by paired (C and D) t test.