CAP350 knockdown leads to defective cystogenesis.

<p>(<b>A</b>) Sequential confocal z sections of a control MDCKII cyst in a 3-D culture from the top (left) to the middle (right) of the cyst. MDCKII cells were plated in matrigel for four days to allow cystogenesis, and cysts were fixed and double labelled for CAP350 and FOP. Single staining with CAP350g is shown. Magnifications at the right of the outlined area show inverted images of CAP350g and FOP labelling. (<b>B</b>) Images showing sequential cross sections of a representative MDCKII control cyst stained for GP135 (blue) as an apical marker, α-catenin (red) as a basolateral marker, and CAP350 (green). Enlarged views of the boxed areas are shown on the lower panels. Note increasing accumulation of CAP350 towards the basal pole of cells (yellow arrows). (<b>C</b>) Box-and-whisker plot showing quantification of diameter in control or CAP350-knockdown MDCKII cysts. Top and bottom end boxes represent 75th and 25th percentiles, and whiskers represent 90th and 10th percentiles. The black line within the box marks the median. **** <i>p</i> < 0.0001 (two-tailed unpaired Student's <i>t</i> test). Data were collected from three independent experiments. (<b>D</b>) Single confocal sections through the middle region of either control (shm4) or CAP350-knockdown (shCAP) cysts fixed at day 4 and labelled for FOP, CAP350, and vinculin. High magnifications of boxed areas are shown at the far-right panels as indicated. (<b>E</b>) Quantification of lumen formation in both control and CAP350-knockdown cysts. Data come from four independent experiments. (<b>F</b>) Single confocal sections through the middle region of either the control (top) or CAP350-knockdown (bottom) cysts labelled for β-catenin, α-catenin, GP135, and ZO-1 as indicated. Rhodamine-phalloidin was used to label filamentous actin (F-actin). DNA was counterstained with 4 ',6-diamino-2-fenilindol (DAPI). Bars = 10 μm. The data used to make this figure are available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002087#pbio.1002087.s001" target="_blank">S1 Data</a>.