Fig 2: Treatment of CTC-Derived Cell Lines with Digitoxin and Ouabain, Related to Figure 6(A) Mean CTC cluster size (area in µm2) of BR16, BRx50, BRx07 and BRx68 CTC-derived cells treated with five different concentrations (1, 5, 10, 20 and 50 nM) of digitoxin or ouabain (n = 4; Error bars represent SEM). Cells that were not treated with FDA-approved compounds (red) or that were filtered (green) are shown for comparison (top). Heatmaps showing the number of nuclei, the average TMRM intensity and the percent (%) of viability of CTC-derived cells treated with digitoxin or ouabain at the indicated concentrations (bottom).(B and C) The histograms show the number of CTC cluster hypomethylated regions that become differentially methylated upon treatment of BR16 (B) and BRx50 (C) cells with 20 nM dixitoxin or ouabain (q = 0.05). Regions that gain >40% methylation are shown in red.(D) Regulatory network analysis of the genes (orange) that are commonly upregulated (log2 fold-change = 0.5) in BR16 and BRx50 CTC-derived cell lines upon 17-day treatment with digitoxin or ouabain, showing TF dependence on PBX3, MAX, NFYA, ELF2, PARP1 and HOXA10. Upregulated genes that do not show dependence on the above TFs are shown below in red.(E) The plot shows the mean cell intensity of Ca2+-bound Fluo-3 after 30 min treatment of BR16 or BRx50 cells with 0.1, 0.5, 1 or 5 µM digitoxin or ouabain, respectively, relative to the untreated control (red) (n = 4; Error bars represent SEM).(F) Western blot for CLDN3, CLDN4 and GAPDH on BR16 cells with double knockout (2KO) of CLDN3 and CLDN4. The dashed line represents the point where irrelevant lanes were spliced out from the original scan.(G) Plot showing the reduction in mean cluster size (area in µm2) of the CLDN3/4 double KO BR16 cells, relative to control BR16 cells. *p < 0.05; **p < 0.01 by Student’s t test. Error bars represent SEM.

Fig 3: Change of contact areas before and after HPI in pH 5.5 (A) and its visualization and quantification by transmission electron microscopy (B). Whereas the combination of oocytes expressing CLDN18 + CLDN18, and CLDN4/18 + CLDN4/18 maintained a larger contact area vs. ctrl after HPI. The difference between CLDN4/18 + ctrl and ctrl pairs was not significantly altered (n = 25–60, Kruskal–Wallis test followed by a Dunn–Bonferroni correction. Representative images; scale bars = 100 µm).

Fig 4: DSP expression alters the cellular response to injury.(A) Cells were plated in 2D and allowed to reach confluence before a scratch wound was made. Wound closure was calculated as a percentage of the initial wound over a 24-hour period. n = 3 experimental replicates of independent wells of a differentiation. (B) Cells were treated with TGF-ß1 (1 ng/ml) for 48 hours before collecting RNA, and COL1A1 expression was measured by qRT-PCR. n = 3 experimental replicates of independent wells of a differentiation. (C) Cells were plated at ALI and then exposed to 5% cigarette smoke in a gas phase or humidified air. Cells were collected 2 hours after smoke exposure, and CLDN4, TJP1, and TJP3 were measured by qRT-PCR. n = 3 experimental replicates of independent ALIs of a differentiation. (D) Cells were exposed to 5% cigarette smoke in a gas phase or humidified air while plated at ALI and then replated in 3D Matrigel to reform alveolospheres over 2 weeks. Colony-forming efficiency was calculated relative to air-exposed cells. n = 3 experimental replicates of independent wells of two differentiations. All error bars represent SD. Statistical significance was determined using unpaired, two-tailed Student’s t test or a one-way ANOVA with a Tukey multiple comparison test; *P < 0.05, **P < 0.005, and ***P < 0.001.

Fig 5: Organ-specific localization of CLDN1, 2, 4 and 5 in canine duodenum, lung, liver and kidney, as detected by immunohistochemistry. (A) CLDN1, (B) CLDN2, (C) CLDN4 and (D) CLDN5 were detected only in the kidney. Boxes in the upper panels (magnification, ×200) are areas magnified in the lower panels (magnification, ×400). Organs for which no boxes are indicated are those that revealed no detectable staining. Arrows indicate positive staining.