Relationship between baseline voltage and response of TER after amiloride addition.

<p>Logarithm of baseline V plotted against ratio of TER upon amiloride addition in drug regime I, applying the initial baseline (A-I), or the rolling baseline approach (B-I). In drug regime II (A-II), grouping produced a significant difference between the two groups (LOW, n = 57; HIGH, n = 24). For both drug regimes, grouping was performed as in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149550#pone.0149550.g005" target="_blank">5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149550#pone.0149550.g006" target="_blank">6</a>. Additional regression and statistical analysis data are shown in Table D, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149550#pone.0149550.s008" target="_blank">S1 File</a>.</p

Human nasal epithelia <i>in vitro</i> reconstitution.

<p>(A) Schematic representation of cell culture protocol for expansion and differentiation of HNEs for Ussing Chamber experiments. (B) Typical HNE monolayer obtained after 7–10 days in culture on collagen-coated flasks. (C) Immunofluorescence of ALI differentiated epithelia: representative acetylated tubulin (green) and ZO-1 (red) with DAPI counterstain (blue) performed after Ussing chamber experiment; 3D reconstruction from z-stack in adjacent left and top panel.</p

Electrophysiological values on an individual donor basis (A-R).

<p>Box plots per donor highlighting minimum, maximum (whiskers) and 25^th/75^th percentile and median (box). Donors arranged in ascending order ranked by mean values of TER in (B) with number of ALIs per donor above the boxes. These are compared with either (A) voltage, (C) [I_eq] and (D) time at ALI.</p

Drug induced effect on TER with respect to the rolling baseline.

<p>TER values at each stage of the drug regime chronologically are considered as baseline. The hypotenuse of the shaded triangular area represents the line of identity while magnification is shown in the box inserts. (A-I) The initial baseline TER values (closed grey circles) are plotted against the values obtained after addition of forskolin (n = 44). (B-I) TER values +FSK are plotted against values after addition of CFTR_Inh172 (n = 44). (C-I) Values of TER with CFTR_Inh172 plotted against values +AMI (n = 44); line of regression: dashed line +99% CI (grey vertical bars). In D-I same data as in C-I showing two distinct populations of amiloride responders designated as LOW (closed grey circles-black dashed regression line +99% CI vertical bars, n = 35) and HIGH (open squares, black regression line +99% CI dotted line, n = 9). (A-II) The initial baseline TER values are plotted against the values obtained after addition of amiloride showing two distinct populations designated as LOW (n = 57) and HIGH (n = 24) amiloride responders. (B-II) Amiloride values plotted against values +FSK (LOW n = 56, HIGH n = 24). (C-II) Values after addition of forskolin plotted against values +CFTR_Inh172 (LOW n = 56, HIGH n = 21). Additional regression and statistical analysis data are shown in Table C in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149550#pone.0149550.s008" target="_blank">S1 File</a>.</p

Donor-level analysis of TER ratio change upon drug addition.

<p>(A-I, A-II) Donors averaged ratios of TER with drug regime I (N = 13) and II (N = 18) respectively, applying the initial baseline approach highlighting the cumulative effect of the drugs. (B-I, B-II) Same data analysed applying the rolling baseline approach to the TER mean values of the individual donors. Error bars: mean ± 95% CI.</p

Baseline electrophysiological values from Ussing chamber experiments.

<p>(A-C). Box plots showing baseline electrophysiological values. Minimum, maximum (whiskers) and 25^th/75^th percentile, median (box line) and mean (cross) are highlighted. (D) Plot of initial V against TER. (E) Table of basic statistical analysis of the data shown in A-C.</p

Ussing chamber ion transport analysis.

<p>(A-I, A-II) Two different drug regimes (I and II) were used and typical representative traces from two donors each are shown. (B-I, C-I) TER values obtained applying drug regime I with their Table of basic statistical analysis. Similarly (B-II, C-II) show the data obtained applying drug regime II. Error bars: mean ± SD; Kruskal-Wallis ANOVA + Dunn’s multiple comparison test: *P<0.05; **P<0.01, ***P<0.001.</p