The effect of DP on the morphological characteristics and the viability of SW620 cells.

<p>A, Morphological changes of SW620 cells observed under phase-contrast microscopy (100 × magnification) after treating cells with (a) control, (b) 5 μM, (c) 10 μM, (d) 20 μM, (e) 40 μM or (f) 80 μM of DP for 24 h. B, SW620 cells were treated with various concentrations (0, 5, 10, 20 40 and 80 μM) of DP for 24 or 48 h, as described in the methods. Cell viability was measured using the MTT assay. The results are expressed as the mean ± S.D. (n = 3); * p < 0.05.</p

Effects of DP on TMEM16A expression.

<p>TMEM16A protein expression were determined by western blotting after SW620 cells were treated with 5 μM of DP for 24, 48 or 72 h (above). Control SW620 cells were treated with DMSO for 24 h. β-actin was used as a loading control. Representative western blots are shown. The bar graph summarizes the relative expression level of TMEM16A protein (below). Expression of TMEM16A protein was normalized with the expression levels of β-actin. All data are shown as the mean ± SD. n = 3; * p < 0.05, ** p < 0.01.</p

Effects of DP on SW620 cell motility.

<p>Migration of SW620 cells was assessed by a wound-healing assay in the presence of 5 μM DP, compared to the control group. Representative images of wound closure were taken at 0, 24, 48 and 72 h after injury under 40 × magnification (above). Bar graphs of wound area are shown (below). Values are the means ± SD; n = 3; ** p < 0.01. Ctrl, control.</p

Effects of DP on SW620 cell migration and invasion <i>in vitro</i>.

<p>Migration (without Matrigel) and invasion (with Matrigel) of SW620 cells are significantly suppressed by 5 μM DP compared with the control group through transwell penetration assays. Non-migrated cells were scraped with a cotton swab. Cells that penetrated the transwell filters were stained with Coomassie blue. Representative images are shown (above). Bar graphs showed cells number per field of SW620 cell migration (lower left) or invasion (lower right). All data are shown as the mean ± SD. n = 3; ** p < 0.01. Ctrl, control.</p

Identification of a small molecule inhibitor (DP) of human TMEM16A.

<p>A, FRT cells stably transfected with human TMEM16A and GFP showing green membrane fluorescence (above) and TMEM16A protein by western blotting (below). B, Examples of whole-cell currents recorded from FRT-TMEM16A cells at a holding potential of 0 mV, followed by pulsing voltages between ±100 mV in steps of 20 mV in the absence (above) or presence of 50 μM DP (below). TMEM16A CaCC currents were elicited by 600 nM of free calcium in pipette solution. C, Current/voltage (I/V) plot of the mean currents at the middle of each voltage pulse. D, Chemical structure of DP. E, CFTR Cl^- current trace recorded from CFTR-expressing FRT cells. CFTR Cl^- current was elicited by 1 mM ATP, followed by the addition of DP and CFTR_inh-172. The dashed line represents zero current. F, The bars represent the percentage inhibition of DP (n = 6) and CFTR_inh-172 (n = 4) on CFTR Cl^- current.</p

Effect of DP on SW480 cell migration and invasion <i>in vitro</i>.

<p>A, Wound-healing assay showed that 5 μM DP did not affect movement of SW480 cells. Representative images of wound closure were taken at 0, 24, 48 and 72 h after injury under 40 × magnification (above). Bar graphs of upper panel are shown (below). Values are the means ± SD; n = 3. Ctrl, control. B, Transwell penetration assays showed that 5 μM DP did not affect migration (without Matrigel) and invasion (with Matrigel) of SW480 cells compared with the control group (above). Bar graphs showed the cell number per field of SW480 cell migration (middle) or invasion (below). Values are the means ± SD; n = 3; Ctrl, control.</p