Figure 6.
CEP120 is essential for HYLS1 recruitment to the centriole. (A) Representative images of HYLS1 localization at the centrioles. Immunofluorescence analysis of centrioles (CEP135), procentrioles (SAS-6), and HYLS1-HA in HYLS1+/+-HA and HYLS1DG/DG-HA RPE1 cells in G1-phase, S/G2-phase, and mitosis. (B) Quantification of immunofluorescence analysis of HYLS1-HA abundance at centrioles (CEP135) of interphase or mitotic HYLS1+/+-HA and HYLS1DG/DG-HA RPE1 cells. (C) Quantification of immunofluorescence analysis of HYLS1-HA abundance at centrioles (CEP135) in serum-starved HYLS1+/+-HA and HYLS1DG/DG-HA RPE1 cells. (D) Representative immunofluorescence images of HYLS1-HA, centrioles (CEP135), and cilia (Ac-TUBULIN) in serum-starved HYLS1+/+-HA RPE1 cells. (E) Representative U-ExM images of centrioles (Ac-TUBULIN), HYLS1-HA, and distal appendages (CEP164) in HYLS1+/+-HA and HYLS1DG/DG-HA RPE1 cells. (F) Representative immunofluorescence images of HYLS1-mNG at the centriole (CEP135) upon CEP120 depletion by siRNA in HYLS1−/− DLD1 cells with HYLS1-mNG (WT or D211G) add-back. Cells were induced with doxycycline for 4 days and CEP120 siRNA transfected on the second day for 48 h. (G) Quantification of HYLS1-mNG intensity at the centriole upon CEP120 depletion by siRNA in HYLS1−/− DLD1 cells with HYLS1-mNG (WT or D211G) add-back. Cells were induced with doxycycline for 4 days and CEP120 siRNA transfected on the second day for 48 h. Data from n = 3 biological replicates were analyzed. (H) Representative U-ExM images of centrioles (Ac-TUBULIN), HYLS1-HA, and CEP120 of HYLS1+/+-HA and HYLS1DG/DG-HA RPE1 cells. (I) Quantification of CEP120 at centrioles (CEP135) of HYLS1+/+ and HYLS1−/− DLD1 cells with HYLS1-mNG (WT or D211G) add-back. Data from n = 3 biological replicates were analyzed. Data are represented as mean ± SEM. Statistical significance was assessed using one-way ANOVA with Tukey’s multiple comparisons test of mean values from each replicate in B, C, G, and I. Only significant results are indicated. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001. Scale bar is 5 µm in A, D, and F and 250 nm in E and H. Inset diameter is 4.4 µm in A, 4.8 µm in D, and 4.3 µm in F. Refer to the image caption for details.

CEP120 is essential for HYLS1 recruitment to the centriole. (A) Representative images of HYLS1 localization at the centrioles. Immunofluorescence analysis of centrioles (CEP135), procentrioles (SAS-6), and HYLS1-HA in HYLS1+/+-HA and HYLS1DG/DG-HA RPE1 cells in G1-phase, S/G2-phase, and mitosis. (B) Quantification of immunofluorescence analysis of HYLS1-HA abundance at centrioles (CEP135) of interphase or mitotic HYLS1+/+-HA and HYLS1DG/DG-HA RPE1 cells. (C) Quantification of immunofluorescence analysis of HYLS1-HA abundance at centrioles (CEP135) in serum-starved HYLS1+/+-HA and HYLS1DG/DG-HA RPE1 cells. (D) Representative immunofluorescence images of HYLS1-HA, centrioles (CEP135), and cilia (Ac-TUBULIN) in serum-starved HYLS1+/+-HA RPE1 cells. (E) Representative U-ExM images of centrioles (Ac-TUBULIN), HYLS1-HA, and distal appendages (CEP164) in HYLS1+/+-HA and HYLS1DG/DG-HA RPE1 cells. (F) Representative immunofluorescence images of HYLS1-mNG at the centriole (CEP135) upon CEP120 depletion by siRNA in HYLS1−/− DLD1 cells with HYLS1-mNG (WT or D211G) add-back. Cells were induced with doxycycline for 4 days and CEP120 siRNA transfected on the second day for 48 h. (G) Quantification of HYLS1-mNG intensity at the centriole upon CEP120 depletion by siRNA in HYLS1−/− DLD1 cells with HYLS1-mNG (WT or D211G) add-back. Cells were induced with doxycycline for 4 days and CEP120 siRNA transfected on the second day for 48 h. Data from n = 3 biological replicates were analyzed. (H) Representative U-ExM images of centrioles (Ac-TUBULIN), HYLS1-HA, and CEP120 of HYLS1+/+-HA and HYLS1DG/DG-HA RPE1 cells. (I) Quantification of CEP120 at centrioles (CEP135) of HYLS1+/+ and HYLS1−/− DLD1 cells with HYLS1-mNG (WT or D211G) add-back. Data from n = 3 biological replicates were analyzed. Data are represented as mean ± SEM. Statistical significance was assessed using one-way ANOVA with Tukey’s multiple comparisons test of mean values from each replicate in B, C, G, and I. Only significant results are indicated. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001. Scale bar is 5 µm in A, D, and F and 250 nm in E and H. Inset diameter is 4.4 µm in A, 4.8 µm in D, and 4.3 µm in F.

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