JPEG Sequences for Scratch Wound experiment

Red, green and ratio immunofluorescence images captured during primary scratch wounding experiment described in paper

ATP_Exp_ JPEG_Sequence

Red, Green and Ratio Jpeg images forming primary dataset for ATP experiment described in paper

Effect of dominant-negative mutant E-cadherin on expression and localisation of endogenous E-cadherin and catenins in NHU cells.

<p>(A) NHU cells expressing the H-2K^d-E-cad mutant (NHU-ECmut) and their isogenic controls (NHU-Con) were established by retrovirus transduction and expression of mutant E-cadherin was assessed by flow cytometry and immunofluorescence microscopy. For flow cytometry (upper panels), expression was analysed using FITC-conjugated anti-H-2K^d antibody (open histograms) alongside irrelevant isotype-matched control antibody (filled histograms). Histograms represent log₁₀ fluorescence intensity in the FL-1 channel. For immunofluorescence microscopy (lower panels), expression was detected using anti-H-2K^d antibody followed by goat anti-mouse antibody conjugated with Alexa Fluor 488 (green). (B) Following initial seeding, NHU cells were cultured in medium containing low (0.09 mM) or physiological (2.0 mM) Ca²⁺ concentrations for 24 hours before expression of E-cadherin, α-catenin and β-catenin was assessed by microscopy using mouse (E-cadherin) and rabbit (catenins) antibodies, followed by goat antisera conjugated with Alexa Fluor 488 (green) or 594 (red). (C) NHU-Con and NHU-ECmut cells were cultured in medium containing physiological [Ca²⁺] and expression of E-cadherin, α-catenin and β-catenin was determined by labelling using primary antibodies above, followed by Alexa Fluor 488-conjugated antibody (green). In all immunofluorescence microscopy experiments, cell nuclei were visualised by labelling with Hoechst 33258 (blue).</p

Loss of β-catenin by shRNA-mediated knock-down mimics E-cadherin engagement.

<p>(A) NHU cells expressing β-catenin shRNA (NHU-β-cat-KD) and their isogenic controls expressing scramble shRNA control (NHU-Con) were established by retrovirus transduction and the efficiency of the shRNA to down-regulate β-catenin was assessed by immunoblotting. Lysates from NHU-Con and NHU-β-cat-KD cells maintained in low calcium were probed for expression of active β-catenin (β-cat), E-cadherin (E-cad), phospho-AKT at S437 (p-AKT) and β-actin using mouse antibodies (β-cat, E-cad and β-actin) and rabbit antiserum (p-AKT), followed by goat anti-mouse antibody conjugated with Alexa Fluor 680 or goat anti-rabbit antibody conjugated with IRDye 800 as appropriate. Densitometry was performed to determine differences in protein expression with respect to β-actin and fold differences following normalisation are indicated. (B) NHU-Con and NHU-β-cat-KD cell lines were seeded into 96-well plates and cultured for a period of 6 days in medium containing either 0.09 mM (Low Ca) or 2.0 mM (Phys Ca) calcium concentration and in the absence (Control) or presence of 5 µM of PI3-K inhibitor LY294002. Proliferation was determined on the basis of cell biomass using the MTT assay and results are presented in upper and lower left panels. Data points represent mean absorbance values for 6 replicate wells (±S.E.M.). To allow easier comparison between inhibitor-treated (+ LY) and non-treated cells, graphs for each cell line are also presented (upper and lower middle panels). In addition, results for all data points are shown in the form of bar graphs (lower panel) for the purpose of statistical analysis. ns, non-significant; *, P<0.05; **, P<0.01; ***, P<0.001.</p

Physiological calcium causes a transient increase in proliferation in low density NHU cultures which is independent of EGFR/ERK signaling.

<p>(A) NHU cells were seeded in 6-well plates at 2×10⁴/well in medium containing 0.09 mM (Low Ca²⁺) or 2.0 mM (Phys Ca²⁺) extracellular [Ca²⁺]. Cell growth was determined by cell counting for a total period of 9 days. Each data point represents the mean (±S.E.M.) of 3 replicates (n = 3) and results are representative of at least three independent experiments. ns, non-significant; *, P<0.05; **, P<0.01. (B) NHU cells were cultured as above and expression of EGFR, phospho-ERK (pERK) and total ERK was assessed by immunofluorescence microscopy using rabbit (EGFR and p-ERK) and mouse (total ERK) antibodies, followed by goat antisera conjugated with Alexa Fluor 488 (green) or 594 (red). Cell nuclei were visualised using Hoechst 33258 (blue). (C) NHU cells were seeded in standard culture medium (0.09 mM calcium) and left to attach overnight. Calcium concentration was then increased to 2 mM and protein lysates were prepared at the indicated time-points for gel-electrophoresis and immunoblotting. Expression of EGFR, phospho-ERK (pERK), total ERK and β-actin was determined using mouse antibodies (total ERK/β-actin) and rabbit antisera (EGFR/pERK), followed by goat anti-mouse antibody conjugated with Alexa Fluor 680 (red) or goat anti-rabbit antibody conjugated with IRDye 800 (green). Densitometry was performed to determine fold induction of EGFR and pERK expression following normalisation against β-actin and total ERK, respectively. Bar graphs represent results relative to the expression level of the initial time point (0 hours).</p

Loss of E-cadherin function enhances NHU cell proliferation and activates the EGFR/ERK and β-catenin/TCF signalling pathways.

<p>(A) NHU-Con and NHU-ECmut cells were seeded in 96-well plates and cultured as above. [³H]-thymidine (TdR) precursor was added 24 hours later and 16 hours post-pulsing, cells were harvested and TdR uptake measured by scintillation spectrometry. Data represent mean of cpm counts (±S.E.M.) for 12 replicate wells. ns, non-significant; *, P<0.05; **, P<0.01. Of note, NHU-Con cells showed higher proliferation levels in low calcium compared to physiologic calcium conditions, hence there was no apparent calcium-mediated enhancement in growth described above. This is because [³H]-thymidine incorporation studies assessed proliferation in NHU cultures that were at >50% confluence, thus missing the initial phase where cells exhibit calcium-mediated increased growth at low-density. (B) NHU-Con and NHU-ECmut cells were cultured in medium containing 0.09 mM (low Ca) or 2.0 mM (phys Ca) [Ca²⁺] and proliferation assessed by cell counting as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013621#pone-0013621-g001" target="_blank">Figure 1A</a>. Each data point represents the mean (±S.E.M.) of 3 replicates and results are representative of at least two independent experiments. Results for days 3, 5 and 7 are also presented in the form of bar graphs (lower panel) for the purpose of statistical analysis and clarity. ns, non-significant; *, P<0.05; **, P<0.01; ***, P<0.001. (C) NHU-Con and NHU-ECmut cells were cultured in medium containing low (-Ca²⁺) and physiological (+Ca²⁺) calcium levels for 24 hours and protein lysates were prepared. Expression of phospho-ERK (p-ERK) and -AKT (p-AKT) as well as total ERK and AKT was determined using primary and secondary antibodies described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013621#pone-0013621-g001" target="_blank">Figures 1C</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013621#pone-0013621-g002" target="_blank">2A</a>. Densitometry was performed to determine fold induction of p-ERK and p-AKT expression following normalisation against total ERK and total AKT, respectively. Bar graphs represent results relative to the expression level for NHU-Con in low calcium (-Ca²⁺). (D) NHU-Con and NHU-ECmut cells were transfected with the TCF/LEF firefly luciferase reporter TOPflash or the FOPflash control plasmid, alongside the Renilla luciferase expression vector pRL-tk. Four hours post-transfection, culture supernatants were adjusted for the desired level of calcium concentration, i.e. 0.09 mM (Low Ca²⁺) or 2.0 mM (Phys Ca²⁺). Cell lysates were prepared and reporter activity assessed in a 96-well format using the Dual-Luciferase Reporter Assay on a microplate reader for pair-wise detection of luminescence activity for each reporter. Firefly luciferase values were normalised against those of Renilla luciferase and were then expressed as fold activity with respect to that obtained for FOPflash in NHU-Con cells cultured in low or physiological calcium. Bars represent mean fold Firefly luciferase activity (±S.E.M.) for 6 replicate samples.</p

Calcium-induced increase in proliferation in low-density NHU cultures occurs via activation of the PI3-K/AKT pathway.

<p>(A) NHU cells were seeded in medium containing low [Ca²⁺] (0.09 mM) and left to attach overnight. Calcium concentration was then increased to 2 mM and protein lysates were prepared at the indicated time-points for immunoblotting. Expression of phospho-AKT (green) and total AKT (red) was determined using rabbit and mouse antibodies, respectively, followed by fluorochrome-conjugated secondary antisera as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013621#pone-0013621-g001" target="_blank">Figure 1C</a>. Immunolabelling was visualised and densitometry to determine fold induction of p-AKT expression with respect to total AKT was performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013621#pone-0013621-g001" target="_blank">Figure 1C</a>. (B) NHU cells were cultured in medium containing 0.09 mM (Low Ca²⁺) or 2.0 mM (Phys Ca²⁺) [Ca²⁺]. Expression of phospho-AKT (p-AKT) was determined by immunofluorescence microscopy using anti-p-AKT rabbit antibody followed by goat anti-rabbit antibody conjugated with Alexa Fluor 488 (green). Cell nuclei were visualised using Hoechst 33258 (blue). (C) NHU cells were seeded into 96-well plates and cultured for a period of 7 days in medium containing either low (-Ca) or physiological (+Ca) calcium levels, in the presence or absence of 5 µM of the PI3-K inhibitor LY294002. Proliferation was determined on the basis of cell biomass using the MTT assay. Data points represent mean absorbance values for 6 replicate wells (±S.E.M.). Results for all data points are also presented in the form of bar graphs (lower panel) for the purpose of statistical analysis. ns, non-significant; **, P<0.01; ***, P<0.001.</p

TER and water and urea permeability coefficients of non-differentiated monolayer cultures and differentiated urothelial constructs established on Snapwell™ membranes.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045339#s2" target="_blank">Results</a> are displayed as mean ± s.d.</p>(1)<p>P_D urothelium after correction for the P_D of the Snapwell™ membrane.</p>(2)<p>P_D significantly different for cultures maintained in non-differentiated versus differentiated states (p<0.0001; unpaired 2-tailed t test).</p>(3)<p>P_D significantly different for cultures maintained in non-differentiated versus differentiated states (p<0.0001; unpaired 2-tailed t test with Welch correction).</p

Immunolocalisation and expression of AQP3 in response to altered medium osmolality.

<p>a) Immunofluorescence labelling of non-differentiated (proliferative) and differentiated NHU cell cultures grown on glass slides showing increased labelling intensity and membrane localisation of AQP3 at high concentrations of NaCl, but not urea, following 72 hours of exposure. Scale bar: 10 µM. b) Immunohistochemistry of differentiated NHU cell constructs grown on permeable Snapwell membranes and exposed for 72 hours to indicated osmolalities. Note no effect of urea, but major increase in AQP3 expression in all layers in response to medium made hyperosmotic (500 mosm/kg) by the addition of NaCl, irrespective of exposure via apical or basal aspect. Scale bar: 50 µM.</p

Water and urea permeability of differentiated urothelial tissue constructs.

<p>Diffusive permeability coefficients across differentiated urothelial cell cultures are shown for (a) [³H]-water and (b) [¹⁴C]-urea. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045339#s2" target="_blank">Results</a> were compiled from a total of 10 urothelial constructs from five independent cell lines. Data shown as mean ± SD.</p