Epithelial cells were stained with Calcein Green and the ASL stained using dextran conjugated Texas Red™ fluorochrome 24 h before estrogen treatment. Panel A shows representative z-plane sections of NuLi-1 (left) and CuFi-1 (right) cells under basal conditions (top) and after 30 mins treatment with 1 nM E2 (bottom). Mean changes in ASL height in control conditions or following treatment with different concentrations of estrogen are shown in panel B for NuLi-1 (solid line) and CuFi-1 (dashed line) cells (n≥4, Error bars reflect standard error of the mean, ANOVA, ** p<0.01, *** p<0.001, compared to control conditions; δ p<0.05, δδ p<0.01, δδδ p<0.001, difference between NuLi-1 and CuFi-1 cells at the indicated estrogen concentration, ns: non significant). Representative z-plane sections of CF primary bronchial epithelial cells under basal conditions (left) and after 30 mins treatment with 1 nM E2 (right) (panel C) and mean changes in ASL height in primary bronchial epithelial cells of 3 CF female children (panel D) (n = 3, paired t-test, ** p<0.01)