Hyls1 D226G and HYLS1 D211G impair distal appendages assembly. (A) Representative images of centrosomes (γ-TUBULIN), distal appendages (CEP164), and cilia (Ac-TUBULIN) in MEFs of Hyls1^+/+, Hyls1^+/DG, and Hyls1^DG/DG animals. (B) Quantification of centrosomes (left), distal appendages (middle), and ciliation frequency (right) from immunofluorescence images of Hyls1^+/+, Hyls1^+/DG, and Hyls1^DG/DG MEFs. MEFs generated across N ≥ 3 mice per genotype were analyzed. (C) Representative images of centrosomes (CEP135), distal appendages (CEP164), and cilia (Ac-TUBULIN) in Hyls1^+/+ and Hyls1^DG/DG RPE1 cells. (D) Quantification of centrosomes (left), distal appendages (middle), and ciliation frequency (right) from immunofluorescence images of Hyls1^+/+ and Hyls1^DG/DG RPE1 cells. Data across n = 3 biological replicates were analyzed. (E) Representative images of centrioles and cilia (Ac-TUBULIN), and distal appendages (CEP164) in MEFs of Hyls1^+/+, Hyls1^+/DG, and Hyls1^DG/DG animals analyzed by U-ExM. (F) Representative images of centrioles and cilia (Ac-TUBULIN) and distal appendages (CEP164) in Hyls1^+/+ and Hyls1^DG/DG RPE1 cells analyzed by U-ExM. Data are represented as mean ± SEM. Foci number and ciliogenesis 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 C and 250 nm in E and F. Inset diameter is 6.9 µm in A and C. Asterisk (*) indicates defective centrioles in F.