(A) Confocal images of live alveoli show TNFR1 expression (red). Images were obtained at baseline (BL) and after indicated alveolar microinfusions (arrows in sketch and plotted data). TNFR1 fluorescence was quantified as gray levels at a region of interest (ROI, dashed circle). The gray levels are shown as the time line plot with the time points of the microinjections marked by arrows. Each plotted point shows the average for 15 ROI from 5 alveoli of a single lung (mean ± SEM). Replicated in 3 lungs. Scale bar: 20 μm. Alv, alveolus. (B) Data show group responses to alveolar microinjection of TNF-α. The response time points are as indicated. For each bar, n = 5. *P < 0.05 versus baseline (dotted line). (C) Confocal images of live alveoli show pseudocolored fluorescence of cytosolic Ca²⁺ and F-actin, as determined by fluorescence of Fluo4 and Lifeact, respectively. The tracings show alveolar epithelial responses to alveolar microinfusion of TNF-α in a single lung. Replicated 5 times in each of 3 lungs. For the tracings, in the baseline image, ROIs were selected at gray levels of 80–120 to accommodate detection of subsequent increases or decreases of fluorescence within the dynamic range of the imaging system. Scale bars: 10 μm. (D and E) Data are whole-image gray levels normalized to the corresponding baseline (dashed lines). Responses shown are to alveolar microinjection of TNF-α after 30 minutes (D). The responses to cytochalasin D (CytD) were obtained after 60 minutes (E). Data are presented as mean ± SEM. Each dot shows data for a single lung. For each bar, n = 4 (C and E) or 5 (D) lungs. *P < 0.05 versus baseline (dotted lines) using ANOVA with Bonferroni correction.