Figure 2.

cep135, wdr8, and ssx2ip KOs generated by CRISPR/Cas9 in human RPE-1 cells line are vital and proliferate. (A) Genomic organization of human CEP135, WDR8, or SSX2IP loci with indicated targeting sites used for CRISPR/Cas9-mediated DNA cleavage. (B) Immunoblot to detect CEP135 (upper panel) or SSX2IP (lower panel) in cell clones after KO of the respective genes. Please note that the SSX2IP antibody tended to reveal unspecific signals in some immunoblots (see Figure 3A). Clones wdr8 ko308, ssx2ip ko2-13, and cep138 ko8 were used for all further experiments. (C) Immunofluorescence to detect CEP135, WDR8, or SSX2IP in human RPE-1 wildtype (WT) and representative cep135, wdr8, or ssx2ip KO cell lines (see Figures S1–S3 for all cell clones). (D) Cell proliferation in WT and wdr8, ssx2ip, and cep135 RPE-1 KO cell lines. A total of 10⁵ cells (3.5 cm cm² dish) were seeded and counted at indicated times (20 h, 32 h, 44 h, and 56 h). (E,F) DNA content analysis using propidium iodide (PI) staining to analyze cell-cycle progression in WT and wdr8, ssx2ip, or cep135 RPE-1 KO cell lines. The indicated range shows cells in S/G₂/M-phase. (F) Quantification of the fraction of cells in S/G₂/M-phase. The graph shows average values from independent experiments ± S.D.; the p-value was calculated using a two-tailed Student’s t-test. * indicates p < 0.05, **** p < 0.001.