Figure 4. Lack of cilia and defects in mother centriole of Cc2d2a-/- MEFs can be rescued by Cc2d2a transgene.
(a) Cc2d2a-/- MEFs have mother centriole but no cilia. Anti-Arl13b is used to highlight cilia. MC (basal body) is stained with γ-tubulin antibody. (b) Cc2d2a transgene rescues cilia biogenesis in Cc2d2a-/- MEFs. Anti-Arl13b staining is shown in red circle. GFP (green) represents the transfected cells. Non-transfected cells in the same field do not have any cilia. Nuclei are stained with DAPI (blue). In c-d cilia are marked by acetylated α– tubulin (green). (c) Pericentrin (a proximal end marker of mother centriole) staining (in red) is unaltered in Cc2d2a-/- MEFs. See also Supplementary Fig. 4. (d) Immunostaining of ninein that marks both SDA and the mother centriole proximal end is significantly reduced in Cc2d2a-/- MEFs. (e) Anti-Odf2 immunostaining which is detected at SDA in wild type is barely visible in Cc2d2a-/- MEFs. (f) Trichoplein immunostaining is reduced in Cc2d2a-/- MEFs. All scale bars are 5 μm. Antibody is indicated above each panel. In merged images, blue nuclear stain is DAPI. The number of cells used for quantitation is indicated in all graphs. The graph in a compares percentage of cells with cilia (y-axis) between wild type (+/+) and Cc2d2a-/- genotypes (x-axis). The graphs in remaining panels compare the level of antibody-generated fluorescence (red signal in each panel) between the two genotypes, where x- and y-axes are genotypes and corrected fluorescence intensity (arbitrary units, (a. u.)), respectively. Bars indicate s.e.m. The sample size (n) is indicated on the bar diagram, and the experiment was repeated three times. P-value was derived from unpaired two tailed t-test. Statistical significance is marked with asterisks (* and **** indicate P≤0.05 and 0.0001, respectively).