HYLS1 is required for centriole microtubule wall stability. (A) Schematic representation of the INDELs introduced by CRISPR in HYLS1^−/− DLD1 cells. (B) Quantification of proximal (CEP135), centriole elongation and structural integrity factors (CPAP, CEP120 and CP110), inner scaffold (POC5), distal end (TALPID3), and distal appendage (ANKRD26) proteins from immunofluorescence images of HYLS1^+/+ and HYLS1^−/− DLD1 cells. Data from n ≥ 1 biological replicates were analyzed. (C) Quantification of centrosome number (CEP135) in HYLS1^+/+, HYLS1^−/−, and HYLS1^−/− DLD1 cells with HYLS1-mNG (WT or D211G) add-back. Data from n ≥ 3 biological replicates were analyzed. (D) Quantification of maximum (left) and minimum (right) centriole length from HYLS1^+/+, HYLS1^−/−, and HYLS1^−/− DLD1 cells with HYLS1-mNG (WT or D211G) add-back. Data from n = 3 biological replicates were analyzed. (E) Schematic representation of the experimental strategy for microtubule stabilization with Taxol in mitosis in HYLS1^+/+ and HYLS1^DG/DG RPE1 cells. (F) Quantification of centriole defects (left), centriole asymmetry, and longest and shortest centriolar microtubule length following Taxol treatment of HYLS1^+/+ and HYLS1^DG/DG RPE1 cells. Data from n = 3 biological replicates were analyzed. (G) Representative U-ExM images of centrioles (Ac-TUBULIN and α/β-TUBULIN) and distal appendages (CEP164) in HYLS1^+/+ and HYLS1^DG/DG RPE1 cells treated with DMSO or Taxol. Data are represented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test from mean values from each replicate (D). (*) 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 data. Scale bar is 250 nm in G. Asterisk (*) indicates defective centrioles.