Figure 2.

KO of genes identified by screening in HEK293 cells. (A) Genes identified in a screening for GPI-APs’ resistance to PIPLC were knocked out by the CRISPR-Cas9 system. KO of the genes is confirmed in Fig. S1. The KO cells were treated with or without PIPLC, stained with anti-CD59 antibody, and analyzed by flow cytometry. Shaded areas indicate cells treated with PIPLC, solid lines indicate cells without PIPLC treatment, and dashed lines show background. (B) Percentages of CD59 remaining after PIPLC treatment of the KO cell lines are plotted. Values are means ± SD of three independent measurements, with p-values (two-tailed Student’s t test) shown on the right. (C) Overexpression of human PGAP1 in the gene KO cell lines rescued the phenotype. Human PGAP1 plasmid was transfected into the KO cells. Cells were selected with antibiotics, treated with or without PIPLC, and then stained with anti-CD59 antibody and analyzed by flow cytometry. The incomplete rescue was a result of the presence of cells showing antibiotics resistance but not expressing PGAP1.