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. 2020 Mar 13;11(5):352–365. doi: 10.1007/s13238-020-00699-6

Figure 1.

Figure 1

The expression of CAS9 alone in hESCs promotes DNA DSB damage and activates DNA damage response pathway. (A) Inducible expression of CAS9 in hESCs activated DNA damage response. Lentivirus harboring the inducible CAS9 expression cassette or control empty vector was used to transduce hESCs. The expression of CAS9 was induced with 2 µg/mL doxycycline (Doxy) treatment for 0, 24 h and 48 h. The relative levels of the phosphorylation of p53, H2AX, CHK1 were indicated at the bottom. n = 3. Data are presented as mean value ± SD. *P < 0.05, **P < 0.01. (B) The expression of CAS9 induced the number of the γH2AX foci in hESC. hESCs with CAS9 inducible expression cassette were plated on chamber slides and treated with or without 2 µg/mL doxycycline for 3 days. The γH2AX foci were detected by a confocal microscope. Scale bar, 10 µm. Unpaired t test. n = 20. Data are presented as mean value ± SD. ***P < 0.001. (C) Detection of DNA DSB damage by Comet assay in hESCs after various treatments. CTL, hESCs with empty expression vector treated with 2 µg/mL doxycycline (Doxy), doxorubicin (Dox), Doxy + Dox, hESCs with CAS9 inducible expression vector were treated with 2 µg/mL doxycycline for three days, 0.5 µmol/L Dox for 2 h, 2 µg/mL doxycycline for three days + 0.5µmol/L Dox for 2 h. Unpaired t test. n = 20. Data are presented as mean value ± SD. ***P < 0.001. (D) CAS9 is present in both nucleus and cytoplasm. CTL or CAS9, hESCs with lentiviral empty vector or CAS9 inducible expression cassette were treated with 2 µg/mL doxycycline for 3 days. Cells were fractionated into nuclear and cytoplasmic fractions, and examined for the levels of CAS9 as well as nuclear and cytoplasmic proteins histone H3 and α-tubulin. (E) The expression of p53 target genes in hESCs after the expression of CAS9. CTL, CAS9, hESCs with CAS9 inducible expression vector were treated with or without 2 µg/mL doxycycline for 3 days. n = 3. Data are presented as mean value ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. (F) Cellular proliferation after the expression of CAS9 in hESCs. n = 3. Data are presented as mean value ± SD. ***P < 0.001. (G) The impact of CAS9 expression on the pluripotency of hESCs. The pluripotency makers TRA1-60 and TRA1-81 were analyzed in hESCs with or without CAS9 expression induced by 2 µg/mL Doxy for 4 days