Figure 5.
HYLS1 overexpression leads to over-elongated centrioles. (A) Representative immunofluorescence images of HYLS1-mNG, centrioles (CEP135), and an inner scaffold protein (CENTRIN2) in HYLS1+/+ and HYLS1−/− DLD1 cells with HYLS1-mNG (WT or D211G) add-back. (B) Representative immunofluorescence images of HYLS1-mNG, centrioles (CEP135), and distal appendages (CEP164) in HYLS1+/+ and HYLS1−/− DLD1 cells with HYLS1-mNG (WT or D211G) add-back. (C) Quantification of inner scaffold protein (CENTRIN2) at centrioles (CEP135) from immunofluorescence images of HYLS1+/+ and HYLS1−/− DLD1 cells with HYLS1-mNG (WT or D211G) add-back. Data from n = 3 biological replicates were analyzed. (D) Quantification of distal appendages (CEP164) at centrioles (CEP135) from immunofluorescence images of HYLS1+/+ and HYLS1−/− DLD1 cells with HYLS1-mNG (WT or D211G) add-back. Data from n = 3 biological replicates were analyzed. (E) Representative images of centrioles (Ac-TUBULIN), and centriolar cap protein (CP110) in HYLS1+/+ and HYLS1−/− DLD1 cells with HYLS1-mNG (WT or D211G) add-back analyzed by U-ExM. (F) Representative images of centrioles (Ac-TUBULIN), and distal appendages (CEP164) in HYLS1+/+ and HYLS1−/− DLD1 cells with HYLS1-mNG (WT or D211G) add-back analyzed by U-ExM. (G) Quantification of centriole defects in HYLS1+/+ and HYLS1−/− DLD1 cells with HYLS1-mNG (WT or D211G) add-back analyzed by U-ExM. Data from n = 3 biological replicates were analyzed. (H) Analysis of the centriole asymmetry in HYLS1+/+ and HYLS1−/− cells with HYLS1-mNG (WT or D211G) add-back analyzed by U-ExM. Data from n = 3 biological replicates were analyzed. Data are represented as mean ± SEM. Statistical significance was assessed using one-way ANOVA with Sidak’s multiple comparisons test (H). (*) P < 0.05, (**) P < 0.01, (***) P < 0.001. Only significant results are indicated. Foci number and centriole defects analysis were assessed using two-way ANOVA with post-hoc analysis and results are summarized in supplementary material. Scale bar is 5 µm in A and B and 250 nm in E and F. Inset diameter is 4.3 µm in A and B. Asterisk (*) indicates defective centrioles. Refer to the image caption for details.

HYLS1 overexpression leads to over-elongated centrioles. (A) Representative immunofluorescence images of HYLS1-mNG, centrioles (CEP135), and an inner scaffold protein (CENTRIN2) in HYLS1+/+ and HYLS1−/− DLD1 cells with HYLS1-mNG (WT or D211G) add-back. (B) Representative immunofluorescence images of HYLS1-mNG, centrioles (CEP135), and distal appendages (CEP164) in HYLS1+/+ and HYLS1−/− DLD1 cells with HYLS1-mNG (WT or D211G) add-back. (C) Quantification of inner scaffold protein (CENTRIN2) at centrioles (CEP135) from immunofluorescence images of HYLS1+/+ and HYLS1−/− DLD1 cells with HYLS1-mNG (WT or D211G) add-back. Data from n = 3 biological replicates were analyzed. (D) Quantification of distal appendages (CEP164) at centrioles (CEP135) from immunofluorescence images of HYLS1+/+ and HYLS1−/− DLD1 cells with HYLS1-mNG (WT or D211G) add-back. Data from n = 3 biological replicates were analyzed. (E) Representative images of centrioles (Ac-TUBULIN), and centriolar cap protein (CP110) in HYLS1+/+ and HYLS1−/− DLD1 cells with HYLS1-mNG (WT or D211G) add-back analyzed by U-ExM. (F) Representative images of centrioles (Ac-TUBULIN), and distal appendages (CEP164) in HYLS1+/+ and HYLS1−/− DLD1 cells with HYLS1-mNG (WT or D211G) add-back analyzed by U-ExM. (G) Quantification of centriole defects in HYLS1+/+ and HYLS1−/− DLD1 cells with HYLS1-mNG (WT or D211G) add-back analyzed by U-ExM. Data from n = 3 biological replicates were analyzed. (H) Analysis of the centriole asymmetry in HYLS1+/+ and HYLS1−/− cells with HYLS1-mNG (WT or D211G) add-back analyzed by U-ExM. Data from n = 3 biological replicates were analyzed. Data are represented as mean ± SEM. Statistical significance was assessed using one-way ANOVA with Sidak’s multiple comparisons test (H). (*) P < 0.05, (**) P < 0.01, (***) P < 0.001. Only significant results are indicated. Foci number and centriole defects analysis were assessed using two-way ANOVA with post-hoc analysis and results are summarized in supplementary material. Scale bar is 5 µm in A and B and 250 nm in E and F. Inset diameter is 4.3 µm in A and B. Asterisk (*) indicates defective centrioles.

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