HYLS knockout leads to impaired recruitment of inner scaffold and centriole distal end proteins. (A) Quantification of centriole length from U-ExM images of Hyls1^+/+, Hyls1^+/DG, and Hyls1^DG/DG MEFs. MEFs from N ≥ 3 mice per genotype were analyzed. (B) Quantification of centriole length from U-ExM images of HYLS1^+/+ and HYLS1^DG/DG RPE1 cells. Data from n = 5 biological replicates were analyzed. (C) Representative U-ExM images of centrioles (Ac-TUBULIN) and CENTRIN2 of Hyls1^+/+ and Hyls1^−/− RPE1 cells. (D) Quantification of centrioles (CEP135) from immunofluorescence images of HYLS1^+/+ and HYLS1^DG/DG RPE1 cells. Data from n ≥ 3 biological replicates were analyzed. (E) Quantification of proximal (SAS-6 and CEP135), centriole elongation and structural integrity factors (CPAP, CEP295, CEP120, and CP110), inner scaffold (POC5 and CENTRIN2), distal end (C2CD3 and TALPID3), and distal appendage (CEP164) proteins from immunofluorescence images of HYLS1^+/+ and HYLS1^−/− RPE1 cells. Data from n ≥ 2 biological replicates were analyzed. (F) Quantification of proximal (CEP135), centriole elongation and structural integrity factors (CEP120), inner scaffold (POC5 and CENTRIN2), distal end (C2CD3 and TALPID3), and distal appendage (CEP164) proteins abundance at the centriole from immunofluorescence images of HYLS1^+/+ and HYLS1^−/− RPE1 cells. 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), and an unpaired two-tailed Student’s t test with Welch’s correction (B and F). (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001. Only significant results are indicated. 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 C. Asterisk (*) indicates defective centrioles.