Fig. 4
Drug-induced loss of barrier integrity is observed over time in a concentration-dependent manner. Results shown for staurosporine (a, b, e–g) and aspirin (c, d, h–j). a–d Array of fluorescence micrographs of the gel region showing distribution of the 150 kDa FITC-Dextran (a, c), and 4.4 kDa TRITC-Dextran (b, d) over time and for various compound concentrations; the loss of barrier integrity results in an increased fluorescent signal. Measurements are taken at 1-h intervals up to 12 h, at 16 h, from 24 to 36 h at 1 h interval, and at 48, 53, 60, 72, 82, 96, and 125 h. In between each interval, the OrganoPlate was placed back into the incubator on the interval rocker platform to maintain the perfusion flow. Five technical replicates of each concentration of a compound were measured on a single plate. One well was excluded from further data analysis, because of a pipetting error (marked with “excl” in white). e, h The progression of the loss of barrier function over time is plotted as the ratio between fluorescent signal in apical and basal regions for the various concentrations of staurosporine (e) and aspirin (h), where the plotted line is the mean of five replicate exposures and error bars depict the standard deviation. f, i Kaplan–Meier curves were generated where survival was defined as showing a leakage score below 40%. Overlapping curves were shifted by 1% for clarity purposes. g, j EC50 values are plotted as a function of exposure time. EC50 values were obtained by fitting a concentration-response curve at each time point based on non-linear regression of leakage scores using normalized response and standard slope and were plotted including 95% confidence interval (CI). EC50 values obtained from time points before the first event in the Kaplan–Meier plot, as indicated by a grayed out line, should be interpreted with caution as the curve fit could be dominated by noise rather than biological effect. All shown graphs were derived from data acquired using 150 kDa FITC dextran. Technical replicates are defined as tubes seeded on the same plate and exposed in the same experimental session. Independent full replicate series were run for both staurosporine and aspirin that are displayed in Supplementary Figs. 3 and 4
