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. 2020 Apr 29;219(6):e201904107. doi: 10.1083/jcb.201904107

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© 2020 Pejskova et al.

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Figure 5. — KIF14 function in primary cilia formation is related to interphase. (A–D) hTERT RPE-1 cells were transfected with the indicated siRNA, serum starved (SFM) for 24 h, and analyzed. (A) Quantification of proportion of binucleated cells by microscopy analysis. Treatment with 0.5 µM cytochalasin D (Cyto D) was used as a positive control. (B) Cell cycle distribution analysis of control or KIF14 siRNA-transfected cells with a comparable proportion of cells in G2/M phase. (C) Representative images of detection of ph-H3⁺ cells (green and pointed out with arrows; DNA, blue); scale bar, 50 µm. (D) Quantification of the percentage of ph-H3⁺ cells, where a total of 40,000 nuclei were counted. (E and F) Examination of the effects of prolonged mitosis, induced by 10 µM monastrol for 2–4 h, on primary cilia formation in hTERT RPE-1 cells (please see the scheme of the experiment in E). (F) Monastrol-mediated mitotic arrest and subsequent release into 10 µM (R)-roscovitine (a cyclin-dependent kinase inhibitor) had only a modest effect on primary cilia formation (please compare black columns). Depletion of KIF14 leads to a significantly lower percentage of ciliated cells, both in controls (“leftover”) and cells that experienced temporal mitotic arrest from monastrol treatment. (G–J) hTERT RPE-1 cells were synchronized in G0 by serum starvation (24 h before siRNA transfection), transfected by the indicated siRNA, and serum starved for an additional 48 h before fixation (please see the scheme of the experiment in G). (H) Representative images (ARL13B in green, γ-tubulin in red, and DNA in blue; scale bar, 20 µm) show a significant effect of KIF14 depletion on ciliogenesis independent of mitosis, quantified as a decreased percentage of ciliated cells (I) and cilia length (J). (K–N) Same experimental setup as in G–J, with an additional 20 µM (R)-roscovitine to halt any proliferation before the transfection with siRNA (please see the scheme of the experiment in K, representative images in L; scale bar, 20 µm, and quantifications in M and N). Asterisks or "ns" indicates statistical significance determined using an unpaired t test (A, B, D, F, J, and N) or the Holm–Sidak method (I and M).