HYLS1 knockout phenocopies the HYLS1 ^DG/DG ciliogenesis and distal end assembly defects. (A) Schematic representation of the strategy used to generate the HYLS1^DG/DG RPE1 cells. (B) Abundance of centrosomes (CEP135) and distal appendages (CEP164) in HYLS1^+/+, HYLS1^DG/DG, and HYLS1^−/− RPE1 cells. Data from n = 3 biological replicates were analyzed. (C) Cell cycle profile analysis of HYLS1^+/+, HYLS1^−/−, HYLS1^+/+-HA and HYLS1^DG/DG-HA RPE1 cells. Data from n = 3 biological replicates were analyzed. (D) Schematic representation of the INDELs introduced by CRISPR in HYLS1^−/− RPE1 cells. (E) Quantification of centrosomes (top), distal appendages (middle), and ciliation frequency (bottom) from immunofluorescence images of Hyls1^+/+ and Hyls1^−/− RPE1 cells. Data across n = 3 biological replicates were analyzed. (F) Representative immunofluorescence images of centrosomes (CEP135), distal appendages (CEP164), and cilia (Ac-TUBULIN) in HYLS1^+/+ and HYLS1^−/− RPE1 cells. (G) U-ExM analysis of distal appendages (CEP164) and cilia (Ac-TUBULIN) in HYLS1^+/+ and HYLS1^−/− RPE1 cells. Data are represented as mean ± SEM. Statistical significance was determined using unpaired two-tailed Student’s t test with Welch’s correction (B). Only significant results are indicated. Cell cycle analysis, foci number and percentage of ciliated cells were assessed using two-way ANOVA with post-hoc analysis and results are summarized in supplementary material. (*) P < 0.05, (**) P < 0.01. Scale bar is 5 µm in F and 250 nm in G. Inset diameter is 3.4 µm in F. Asterisk (*) indicates defective centrioles.