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. 2019 Mar 21;104(4):731–737. doi: 10.1016/j.ajhg.2019.02.018

Figure 3.

Figure 3

FAM149B1-Related Ciliopathy Is Associated with Impaired Cilia Structure and Function

(A and B) Immunofluorescence images of the serum-starved fibroblasts from the affected individual in family 1 (A) and control fibroblasts (B) stained for the ciliary marker acetylated α-tubulin (Sigma-Aldrich, T7451) (green) and DNA (blue) showing marked increase in the length of the cilia with normal cilia frequency in the affected individual cells compared to controls. Scale bars represent 10 μm.

(C) Point chart of the ciliary length measured using ImageJ showing significantly longer cilia in the affected individual compared to two control subjects. Bars represent the mean with the SEM, p value < 0.0001.

(D) Immunofluorescence staining images of the affected individual 17DG0722 fibroblast cilia, affected individual 17DG0168 with mutation in TBC1D32, and control stained with the ciliary marker acetylated α-tubulin (Sigma-Aldrich, T7451) (green) and IFT-88 (ProteinTech, 13967-1-AP) (red) (top) or IFT-46 (Abcam ab122422) (red) (bottom) showing marked “pooling” of IFT-88 and IFT-46 at the tip with apparent bulging and bulbous morphology. Scale bars represent 1 μm.

(E) Left column: bar graph showing the relative quantification to the mRNA expression of GLI1 and PTCH1 which were markedly lower in fibroblasts from the affected individual cells 17DG0722 in family 1 than control fibroblasts in response to Smoothened Agonist (SAG) stimulation of 100 nM for 18 h on the basis of two independent experiments (each performed in triplicate). Right column: bar graph showing the relative quantifications to the mRNA expression of GLI1 and PTCH1; the significantly lower expression of GLI1 and PTCH1 and lower induction of GLI1 in response to SAG stimulation in siRNA of FAM149B1 transcript in HEK293 cells in comparison to negative control scrambled siRNA. Quantification was based on three independent samples, each run in triplicate. Error bars represent the SEM, statistical significance represents results from two-tailed Student’s t test and two-way ANOVA are indicated by ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; ns, non-significant.