Figure 2

Stable knockdown of Ift88 or Kif3a inhibits formation of NP cell primary cilia. (a) Ift88 mRNA levels in NP cells transduced with control (ShCtr) or two different ShIft88 clones were measured by qRT-PCR to confirm the knockdown (n ≥ 5). (b) Western blot image showing significant reduction of IFT88 protein levels after the knockdown of Ift88. (c) Densitometry analyses of Western blots confirm significant knockdown of IFT88 (n ≥ 5). (d–f) qRT-PCR, Western blot, and corresponding densitometry analyses, show significant downregulation of KIF3A after stable knockdown using two different ShKif3a clones (n ≥ 4). (g) Acetylated α-tubulin immunofluorescence staining after lentiviral transduction of ShIft88 or ShKif3a shows inhibition of primary cilia formation in majority of rat NP cells. Scale bar = 75 μm. White arrowheads point to primary cilia. (h,i) Quantitation of percentage of NP cells with primary cilia and primary cilium length after stable silencing of Ift88 or Kif3a (n = 3; at least 150 cells/group). Data are represented as scatter plots (mean ± SEM). ns = not significant. One-way ANOVA or Kruskal-Wallis test with Sidak’s, Holm-Sidak’s, or Dunn’s multiple comparison test was used based on the distribution of the data to determine statistical significance. For statistical comparison of the percentages of NP cells with primary cilia, Fisher’s exact test was used. Western blot images were cropped and acquired under same experimental conditions. See Supplementary Fig. S1–1 for un-cropped Western blot images.