Fig. 6

AQP1 deletion does not affect EC differentiation from H1 stem cells but inhibits tube formation of the derived ECs. A, Schematic representation of the epiCRISPR system for AQP1 gene knock-out in H1 cell line. The vector contains a U6 promoter-driven gRNA scaffold, an EF1a promoter-driven Cas9 fused to puromycin-resistance gene and GFP with P2A peptides, and OriP/EBNA1 elements for the plasmid replication in eukaryotes. The epiCRISPR vector can replicate in H1 cells and can be partitioned into daughter cells. B-D, Flow cytometry analyses showing the percentages of cells that express CD31 and CD144 endothelial markers in ECs derived from H1 cell line (B) and two AQP1-knockout H1 cell lines, ΔAQP1-1-H1 (C) and ΔAQP1-2-H1 (D). E-G, Q-PCR analyses confirming the gene expressions of AQP1, CD31, and CD144 in ECs derived from H1 (E), ΔAQP1-1-H1 (F), and ΔAQP1-2-H1 (G) cell lines. H-J, Representative microscopy images showing the tube formation of ECs derived from H1 cell line (H), ΔAQP1-1-H1 (I), and ΔAQP1-2-H1 (J) cell lines. The derived ECs are seeded on the Matrigel for 6 h before being subjected to analysis. The number of junctions, the total length of tubes, and total mesh size of the vascular network formed by ECs derived from H1 (n = 6), ΔAQP1-1-H1 (n = 6), and ΔAQP1-2-H1 (n = 6) cells are shown in K, L, and M, respectively. Data are shown as mean ± s.e.m. One-way ANOVA analysis is applied. ns, not significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bar, 100 μm.