Figure S8.
HYLS1 is asymmetrically localized to the younger parent centriole. (A) Quantification of immunofluorescence analysis of HYLS1-HA abundance at centrioles (CEP135) of interphase or mitotic HYLS1+/+-HA and HYLS1DG/DG-HA RPE1 cells. (B) Representative U-ExM images of cilia (Ac-TUBULIN), distal appendages (CEP164), and HYLS1-HA in HYLS1+/+ and HYLS1DG/DG serum-starved RPE1 cells. (C) Representative U-ExM images of centrioles (Ac-TUBULIN and β-TUBULIN) and HYLS1-HA in HYLS1+/+ DLD1 cells with HYLS1-HA (WT or D211G) add-back. (D) Representative U-ExM images of centrioles (Ac-TUBULIN) and HYLS1-GFP in HYLS1+/+ DLD1 cells expressing HYLS1+/+-EGFP. (E) Quantification of immunofluorescence analysis of SAS-6 and CEP135 abundance at centrioles (CEP135) of interphase or mitotic HYLS1+/+-HA and HYLS1DG/DG-HA RPE1 cells. (F) Quantification of CEP120 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. (G) Quantification of immunofluorescence signal intensity for CEP120 and centrioles (CEP135) in HYLS1−/− DLD1 cells with HYLS1-mNG (WT or D211G) add-back. Data from n = 3 biological replicates were analyzed. (H) Quantification of centriole foci number for HYLS1-mNG, CEP120, and centrioles (CEP135) in HYLS1−/− DLD1 cells with HYLS1-mNG (WT or D211G) add-back. Data from n = 3 biological replicates were analyzed. (I) Representative U-ExM images of centrioles (Ac-TUBULIN), HYLS1-HA, and CEP120 in HYLS1+/+-HA RPE1 cells. (J) Quantification of CPAP 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 determined using one-way ANOVA with Tukey’s multiple comparisons test (A, E–G, and J). (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001. Foci number analysis was assessed using two-way ANOVA with post-hoc analysis and results are summarized in supplementary material. Scale bar is 250 nm in B–D and I. Refer to the image caption for details.

HYLS1 is asymmetrically localized to the younger parent centriole. (A) Quantification of immunofluorescence analysis of HYLS1-HA abundance at centrioles (CEP135) of interphase or mitotic HYLS1+/+-HA and HYLS1DG/DG-HA RPE1 cells. (B) Representative U-ExM images of cilia (Ac-TUBULIN), distal appendages (CEP164), and HYLS1-HA in HYLS1+/+ and HYLS1DG/DG serum-starved RPE1 cells. (C) Representative U-ExM images of centrioles (Ac-TUBULIN and β-TUBULIN) and HYLS1-HA in HYLS1+/+ DLD1 cells with HYLS1-HA (WT or D211G) add-back. (D) Representative U-ExM images of centrioles (Ac-TUBULIN) and HYLS1-GFP in HYLS1+/+ DLD1 cells expressing HYLS1+/+-EGFP. (E) Quantification of immunofluorescence analysis of SAS-6 and CEP135 abundance at centrioles (CEP135) of interphase or mitotic HYLS1+/+-HA and HYLS1DG/DG-HA RPE1 cells. (F) Quantification of CEP120 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. (G) Quantification of immunofluorescence signal intensity for CEP120 and centrioles (CEP135) in HYLS1−/− DLD1 cells with HYLS1-mNG (WT or D211G) add-back. Data from n = 3 biological replicates were analyzed. (H) Quantification of centriole foci number for HYLS1-mNG, CEP120, and centrioles (CEP135) in HYLS1−/− DLD1 cells with HYLS1-mNG (WT or D211G) add-back. Data from n = 3 biological replicates were analyzed. (I) Representative U-ExM images of centrioles (Ac-TUBULIN), HYLS1-HA, and CEP120 in HYLS1+/+-HA RPE1 cells. (J) Quantification of CPAP 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 determined using one-way ANOVA with Tukey’s multiple comparisons test (A, E–G, and J). (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001. Foci number analysis was assessed using two-way ANOVA with post-hoc analysis and results are summarized in supplementary material. Scale bar is 250 nm in B–D and I.

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