Dynamics of CHT trafficking are altered by LY294002 treatment.

<p>Cells were grown for 24 h in the presence of either vehicle or 10 μM LY294002 before being imaged by TIRF microscopy. CHT proteins were fluorescently labeled as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132934#pone.0132934.g003" target="_blank">Fig 3</a>. The imaging protocol was as follows: the dish was set on the stage of the TIRF microscope and imaging was started for 30–60 sec, then a small volume of 2.5 M KCl was applied to the area of the cells by pressure ejection and imaging was continued for a further 3–4 min. Images were captured at 150 nm depth from the coverslip, and experiments were performed at room temperature. <b>Panel A.</b> Representative traces of changes in the cellular fluorescence levels over the time course of imaging with various treatments, as indicated. The arrows indicate when KCl was applied to the cells. <b>Panel B.</b> Area-under-the-curve (AUC) analysis of fluorescence images was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132934#pone.0132934.g003" target="_blank">Fig 3</a>. <b>Panel C.</b> Percentage changes in cell surface fluorescence levels following K⁺-depolarization levels calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132934#pone.0132934.g003" target="_blank">Fig 3</a>. <b>Panel D.</b> Time-to-peak value calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132934#pone.0132934.g003" target="_blank">Fig 3</a>. <b>Panel E.</b> Time taken for cell fluorescence to return to baseline values calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132934#pone.0132934.g003" target="_blank">Fig 3</a>. In these experiments, 1 to 5 regions of interest having high fluorescence corresponding to 1 to 5 individual cells were analyzed per culture dish, with 4 to 5 dishes analyzed per treatment group in 3 independent experiments. Data in Panels B to E are expressed as the mean ± SEM, with statistically-significant differences assessed by unpaired Student’s <i>t</i>-test; asterisks denote <i>p</i> ≤ 0.05.