Fig. 6: Depletion of Pcm1 causes deregulated centriolar targeting of the identified FGM proteins.

Pooled data are presented as mean ± s.d. with sample dots. Two-tailed Mann–Whitney U-test: *P < 0.05; **P < 0.01; ***P < 0.001. mEPCs treated with siRNAs as in Fig. 3a were fixed at day 3 (a, b) or day 5 and 10 (e, g, h) and subjected to 3D-SIM. a, b Representative 3D-SIM images for Cep135 and Cep131. The regions indicated by white arrowheads were magnified to show details. Yellow arrowheads in the color-coded insets point to typical nascent centrioles. The immunofluorescent signals for Centrin were less satisfactory in the four-color immunostaining. Two alternative stages are indicated in cases when we have difficulty to clearly define the precise stage of some cells. c A summary for the indicated proteins. See Supplementary Fig. 5 for 3D-SIM images of additional proteins. d Procentriolar localizations of Cep135, Cep120, and Ofd1 increased upon the depletion of Pcm1. Procentriolar intensities were quantified from three independent experiments and at least five MCCs in each experiment and condition. Note that, although the Pcm1-depleted cells in stages II and III were indistinguishable, the mixed populations lacked procentrioles with weak centriolar Cep135, Cep120, or Ofd1 that resembled those in the stage-II control cells, suggesting a deregulated procentriolar targeting in stage II. e, f Depletion of Pcm1 increased the length of the Cep131 and Centrin double-positive streaks in multiciliated mEPCs. The quantification results (f) were measured from 3D-SIM images (e) collected in two independent experiments. As multicilia are mostly protruded toward the z-axis, length measurement using z-projected images would generate underestimated results. To diminish such underestimation, we only measured five longest Cep131 streaks in each MCC. g The CP-foot was positioned in the axonemal central lumen and aberrantly elongated following time in the Pcm1-depleted mEPCs. Ac-tub and Cep162 labeled axonemal microtubules and transition zone, respectively. Arrows indicate representative long CP-feet labeled with Centrin. Diagrams illustrate distribution patterns of the proteins. h The CP-foot excluded Hydin and partly overlapped with radial spokes. Hydin is associated with CP microtubules. Rsph4 is a radial spoke subunit. Arrows indicate representative cilia. Source data are provided in the Source data file.