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. 2021 Apr 17;296:100680. doi: 10.1016/j.jbc.2021.100680

Figure 1.

Figure 1

DIAPH3 regulates ciliogenesis and cilia length.A–C, impairment of ciliation and cilia length by DIAPH2 depletion cannot be rescued by other isoforms. hTERT-RPE1 cells were transfected with siCtr or siDIAPH2#1 for 48 h, and cells were rescued using wild-type constructs of DIAPH1-myc, DIAPH2-myc, and DIAPH3-myc. A, western blots of samples blotted for human DIAPH2, myc, and GAPDH. Quantifications of (B) ciliation and (C) cilia length are shown. Error bars in B represent ± SD of three independent experiments; n = 50 or more each. Graph of cilia length measurement (C) is displayed in box-and-whisker plot, where the upper and lower quartiles are indicated as the ends of the box, the median is marked by a horizontal line inside the box, mean is marked by a cross sign inside the box, and whiskers are two lines outside the box that extend to the highest and lowest observations, while dots outside this range represent outliers. Two-tailed t-test analysis was done to compare control siRNA to all samples or siDIAPH1 to rescue samples, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, ∗∗∗∗p < 0.001. D–F, reduction of ciliation and cilia length by DIAPH3 depletion cannot be rescued by other isoforms. hTERT-RPE1 cells were transfected with siCtr or siDIAPH3#1, and cells were rescued using wild-type constructs of DIAPH1-myc, DIAPH2-myc, or DIAPH3-myc. D, western blots of rescued cells blotted for human DIAPH3, myc, and GAPDH. E and F, quantification of ciliation and cilia length in hTERT-RPE1 cells treated with siRNA for 48 h, respectively. Error bars in E represent ±SD of three independent experiments; n = 50 or more each. Two-tailed t-test analysis was done to compare control siRNA to all samples or siDIAPH1 to rescue samples, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, ∗∗∗∗p < 0.001. Graph of cilia length measurement is displayed in a box-and-whisker plot as in C. G and H, DIAPH2 and DIAPH3 localize at the base of cilia. hTERT-RPE1 cells were grown to confluence and serum starved for 24 h. Cells were stained with Actub (axonemal marker) and centrin (CNT) for cilia basal marker. Cells were costained for (G) DIAPH2 and CNT or DIAPH2 and Actub. H, cells were costained for DIAPH3 and CNT or DIAPH3 and Actub. Scale bars in G and H are 10 μm in the left panels and 1 μm in the right-side panels.