<i>Azi1</i> knock-down leads to reduced ciliogenesis.

<p>Mouse ShhLIGHT-II fibroblast cells were transfected with siRNA (a non-targeting control siRNA (Ctrl), two positive control siRNAs targeting <i>Ift88</i> (Ift88 #3 and #4) or a pool of four siRNAs targeting the 3′ UTR of <i>Azi1</i> (<i>Azi1</i> 3′UTR)), along with plasmids encoding either <i>GFP</i> or <i>Azi1-GFP</i> (which lacks the 3′UTR of <i>Azi1</i>). (A) Western blot probed with anti-Azi1 antibody (SF91) shows endogenous Azi1 levels are reduced upon <i>Azi1</i> 3′UTR siRNA addition (lower arrow). Tagged Azi1-GFP protein is also detected (upper arrow). The blot was reprobed with anti-α Tubulin as a loading control. Below is quantification of total Azi1 levels (endogenous plus overexpressed), relative to α-Tubulin. (B–E) Cells were stained with anti-Arl13b to mark the cilia and GFP Booster (Chromotek) to enhance the GFP signal. Transfected cells are highlighted with arrowheads; closed arrowheads highlight cells with cilia, open arrowheads highlight cells without cilia. (F) Addition of <i>Azi1</i> 3′UTR siRNA significantly reduced the percentage of cells with cilia, and this reduction is rescued to wild type levels by co-transfection with <i>Azi1-GFP</i>. Shown is the mean +/− SEM of two technical and two biological duplicates (*** <i>P</i><0.001, chi-squared test). Scale bars represent 10 µm.