Fig. 1: High-throughput genetic screening to identify genes sensitive to H₂O₂.

A Phase-contrast image of mouse haESCs used for screening. Scale bar, 100 μm. B Vector designs of the PB-trapping vector and PBase. C Surviving PB-trapping vector-transfected haESC colonies after selection by puromycin for 48 h. HaESCs without transfection were used as controls. Scale bar, 100 μm. D Cell viability of WT-haESCs treated with 0, 0.4, 0.8 and 1.2 mM H₂O₂ for 4 h. Data represent three independent experiments. t-test, ***p < 0.001. Data are presented as the mean ± SD. E Cell viability of haESCs treated with 0.8 mM H₂O₂ for 0, 0.25, 4, and 8 h. Data represented three independent experiments. t-test, ***p < 0.01, ***p < 0.001. Data were presented as the mean ± SD. F Schematic overview of the identification of H₂O₂ toxicity-targeting genes. Surviving cells were analyzed by subsequent sequencing. G DRAQ7 staining analysis of cell viability in Puro^r haESCs and WT-haESCs treated with 0.8 mM H₂O₂ for 4 h. PE-Cy7-positive cells indicated dead cells in the cell cultures. H Phase-contrast images of WT-haESCs (left) and Puro^r haESCs (right) 4 days after being treated with 0.8 mM H₂O₂ for 4 h. Scale bar, 100 μm. I Cell viability analysis of Puro^r haESCs and WT-haESCs 4 days after treatment with 0.8 mM H₂O₂ for 4 h by CCK-8 assay. Data represented three independent experiments. t-test, ***p < 0.001. Data were presented as the mean ± SD.