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Fig. 8. Tight junction (TJ) formation around PAP triggers lumen initiation. (A) Localization of occludin-1 and gp135 in two-cell aggregates with a PAP or with a lumen. In the merged images, grey lines indicate the aggregate outline. Frequencies of aggregates (± s.e.m.) with cytoplasmic (w/o TJ) or ring-like occludin localization surrounding PAP (with TJ; ring in X-Y view or dots in X-Z view; arrowheads) relative to all two-cell aggregates with PAP. Scale bars: 5 µm. (B) Maximum projections of 5-20 central sections of deconvolved confocal images of MDCK two-cell aggregates transiently expressing mRFP-gp135 stained without permeabilization with anti-gp135 antibodies (gp135 ab). Scale bars: 5 µm. (C) Frequencies of aggregates containing PAP and lumen in dependence of mSnail overexpression. The frequencies of control Tet-Off MDCK cells (TO) and two different clones overexpressing mSnail (#1 and #10, ±dox; with/without doxycyclin) in a group of randomly picked two-cell aggregates (n>45) are represented (± s.e.m.; three experiments). Significantly decreased frequencies upon overexpression of mSnail (P<0.05) are indicated with an asterisk. Key for column colours is shown in D. (D) Frequencies of PAP- and lumen-containing aggregates in cultures after discontinuation of mSnail expression. mSnail overexpression was stopped after 40 hours by addition of dox (0 h no mSnail). The mSnail #1 cultures were grown for an additional 4 or 8 hours and quantified as in (C) (4 h and 8 h no mSnail; n>50; three replicates). After 4 hours of release, the cells were exposed for an additional 4 hours to 20°C (20°C) or 0.1% ethanol in medium (EtOH), BFA, bumetanide, or silver nitrate (Ag+). In the negative control `–dox', the cells were grown in dox-free medium for a total of 48 hours; in the positive control `+dox', cells were grown in presence of dox for 48 hours. The sequence of treatments is indicated with a vertical arrow. The categories of aggregates with a statistically significant difference in frequencies in different conditions are connected to the corresponding controls with white bars. The significance is shown with asterisks (*P<0.01; **P<0.001).
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