VAMP7 knockdown decreases cilia frequency and cilia length in MDCK cells.

<p>(A) MDCK 2001 cells were transfected as indicated with control siRNA, our standard VAMP7 siRNA (VAMP7 #1), a less effective VAMP7 targeting sequence (VAMP7 #2), or VAMP8 siRNA. Cells were cultured for four days on permeable supports, then fixed and processed for indirect immunofluorescence to detect acetylated tubulin and cell nuclei. Representative fields acquired using epifluorescence microscopy are shown. Scale bar = 10 µm. (B, D) The number of cells in multiple fields was determined using DAPI nuclear stain and used to quantify the fraction of cells with a primary cilium in each sample. The average of three experiments is plotted (mean +/− SEM); *p<0.05 compared to control using one-way ANOVA with Bonferroni correction. (B) Control n = 1,907; VAMP7 n = 1,747; VAMP7 #2 n = 1,608 cells. (D) Control n = 1,040, VAMP7 n = 1,169, VAMP8 n = 1,049 cells. (C, E) Cilia lengths in multiple fields were measured using ImageJ software, then sorted by length in ascending order and graphed by percentile (100^th percentile = longest cilium in each sample). Each point on the graph represents the length of an individual cilium. (C) There was a statistically significant reduction in cilia length in cells transfected with VAMP7 #1 and VAMP7 #2 siRNAs, as assessed by comparing the medians from four experiments (*p<0.05, **p<0.01 using one-way ANOVA with Bonferroni correction). (E) There was a statistically significant reduction in cilia length in VAMP7 depleted cells, as assessed by comparing the medians from three experiments (*p<0.05 using one-way ANOVA with Bonferroni correction).