HYLS1 is essential for centriole structural integrity and distal end formation. (A) Representative U-ExM images (left) of centrioles (Ac-TUBULIN), quantification of centriole defects (top right), and analysis of the centriole asymmetry (bottom right) in MEFs of Hyls1^+/+, Hyls1^+/DG, and Hyls1^DG/DG animals. MEFs generated across n ≥ 3 mice per genotype were analyzed. (B) Representative U-ExM images (left) of centrioles (Ac-TUBULIN), CENTRIN2, and distal appendages (CEP164), quantification of centriole defects (top right), and analysis of the centriole asymmetry (bottom right) of Hyls1^+/+ and Hyls1^DG/DG RPE1 cells. Data from n = 5 biological replicates were analyzed. (C) Quantification (upper) and representative immunofluorescence images (lower) of centriole proximal marker (SAS-6) and centriole elongation and structural integrity factors (CPAP, CEP295, CEP120, and CP110) in Hyls1^+/+ and Hyls1^DG/DG RPE1 cells. Data from n ≥ 3 biological replicates were analyzed. (D) Quantification (upper) and representative immunofluorescence images (lower) of inner scaffold proteins (POC5 and CENTRIN2), distal end proteins (C2CD3 and TALPID3), and distal appendages (CEP164) in Hyls1^+/+ and Hyls1^DG/DG RPE1 cells. 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 (A – bottom right) and an unpaired two-tailed Student’s t test with Welch’s correction (B – bottom right). (**) P < 0.01, (***) P < 0.001. Only significant results are indicated. Centriole defects or 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 A and B and 5 µm in C and D. Inset diameter is 4.3 µm in C and D. Asterisk (*) indicates defective centrioles.