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. 2021 Jun 25;22(8):e51902. doi: 10.15252/embr.202051902

Figure EV5. Characterization of ciliogenesis and cell cycle phenotypes in cilium assembly and disassembly experiments.

Figure EV5

  1. Quantification of ciliogenesis efficiency and cilium length in control and PCM1‐depleted RPE1 cells treated with DMSO or MLN8237. Cells were transfected with control or PCM1 siRNA#2 for 48 h and treated with DMSO (vehicle control) or 0.5 μM MLN8237 in serum starvation medium for 24 h. Cells were fixed and immunostained for the primary cilium with acetylated tubulin antibody (Acet‐tub) and the centrosome with gamma‐tubulin antibody. n > 100 cells per experiment. Data represent mean value from two experiments per condition ± SEM (***P < 0.001; *P < 0.1; ns, non‐significant, unpaired Student's t‐test).
  2. Quantification of ciliogenesis percentage in RPE1 WT and PCM1 KO cells treated with DMSO (control) and 0.5 μM MLN8237. n > 100 cells per experiment. Data represent mean value from two experiments per condition ± SEM (***P < 0.001, ns: non‐significant, unpaired Student's t‐test).
  3. Representative images and quantification of proliferating cells upon serum starvation. RPE1 cells were transfected with control or PCM1 siRNA for 48 h, serum‐starved for 24 h, and treated with DMSO or 0.5 μM MLN8237. Cells were fixed and immunostained with Ki67 to mark proliferating cells and acetylated tubulin to mark the primary cilium. DNA was stained with DAPI. Scale bar, 10 μm. Ciliated Ki67‐ cells and Ki67‐ cells were quantified. Data represent mean value from two experiments per condition ± SEM (**P < 0.01, ns: non‐significant, unpaired Student's t‐test).
  4. Representative immunofluorescence images for cilium disassembly experiments. RPE1 cells were transfected with control or PCM1 siRNA for 48 h, serum‐starved for 24 h, and treated with DMSO (vehicle control) or 0.5 μM MLN8237 in serum stimulation medium for 2 and 24 h. Cells were fixed and immunostained with antibodies against acetylated tubulin antibody, gamma‐tubulin antibody to mark the centrosome. DNA was stained with DAPI. Scale bar, 10 μm.