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. 2021 Jul 27;13(15):3783. doi: 10.3390/cancers13153783

Figure 4.

Figure 4

EWSR1–FLI1 gene inactivation induced generalized senescence. (a) β-Galactosidase activity was measured in control (Ctrl) cells (A673/sgRNA) and A673/Cas9/sgRNA cells using a β-galactosidase activity assay. β-Galactosidase-positive cells were counted, and the percentage of positive cells were determined. The graphs represent the results of two independent experiments performed with MOI = 1 (1) and MOI = 5 (2). The A673/TR/shEF cells were stimulated with doxycycline (1 µg/mL) for 7 days to induce the expression of the EWSR1–FLI1-specific shRNA (mean ± Scheme 0. Student’s t-test). Representative micrographs of each cell line are also shown. (b) Representative micrographs of A673 and A673/Cas9/FLI1-EX9 cells, showing the characteristic appearance of senescent cells in A673/Cas9/FLI1–EX9 cells. (c) The A673/Cas9/sgRNAs cells were seeded at a low density and then isolated clones were reseeded independently in 96-well plates. Afterward, Sanger sequencing/ICE CRISPR analysis was performed for each clone to determine the gene edition percentage (% indels). The tables show the phenotype of each picked-up clone, if it expand after reseeding and the percentage of gene edition.