Figures & data
Table 1 The number of cells increasing their cytosolic calcium concentration after mechanical stimuli
Figure 1 Method controls for gap junctional and ATP mediated calcium wave blockers. A) Normal urothelial cells were grown on 3 cm diameter dishes and differentiated with 1.8 mM calcium. The cells were loaded with 10 μM calcein and a random cell cluster was selected. One of the cells was bleached with laser and the recovery of the fluorescence was observed at 1-minute intervals the total number of images being 11. The experiment was repeated five times for no treatment and four times for 1-heptanol treatment. B) Normal urothelial cells were seeded on two dishes, loaded with Fluo-3 and 100 μM ATP was added to the cells while changes in calcium concentration were monitored with confocal microscope. One of the dishes was treated with 50 U/ml apyrase prior to the experimentation.
Table 2 The influence of PKC α and βI isoenzyme inhibition on distance, velocity and ▵ intensity of calcium waves
Figure 2 Calcium mediated signal transduction in normal urothelial and 5637 TCC cells with and without Gö6976. A) Upper image is taken prior to micromanipulator stimulation of one cell and lower image immediately after the calcium wave had reached its maximal distance (white arrows). Pseudocolors from black through rainbow colors to red are used to illustrate changes in the intracellular calcium concentration. B) Curves beneath images illustrate changes in cytosolic calcium concentrations within selected cells: black line for stimulated cell and gray line for the most peripheral reacting cell. Black and gray arrowheads indicate the time point where cells start to raise their cytosolic calcium concentration. All images are presented with the same magnification.
Table 3 The length of calcium wave measured in cell rows
Figure 3 Western transfer analysis of connexin expression. A) Western transfer analysis was performed with samples of normal urothelial cells (Normal cells) and 5637 TCC cells (TCC) treated with different concentrations (0 nM, 100 nM, 1000 nM) of Gö6976 for 24 h. Cx26 and Cx32 detection was done with polyclonal and Cx43 with monoclonal antibody. Beta-actin labeling shows equal sample loading. Western transfer analysis was performed with cell lysate from normal cell culture of one patient. B) To study whether Gö6976 treatment affects Cx26 levels in 5637 TCC cells, western transfer analysis was repeated three times and the intensity ratios of Cx26 and Beta-actin bands were determined. Intensity ratios are presented on y-axis and x-axis demonstrates different concentrations of Gö6976. The image presents three different western blot experiments (grey lines) and an average value presented as black line.
Figure 4 Indirect immunofluorescence labeling using monoclonal antibodies against connexins 26, 32, and 43. Panels in the left demonstrate expression of different connexins in normal urothelial cells culture before (−) and after (+) Gö6976 tretament. Panels in the right demonstrate the expression of different connexins in TCC cell culture without (−) and with (+) Gö6976 treatment. Nuclei have been stained with hoechst (blue) and connexin immunosignal is in red. All images are presented with the same magnification. Small boxes within panels show a demonstrative area of each picture in higher magnification. Scale bar 50 μm.
