Figure 3.

The expression of CAS9 in human fibroblasts promotes DNA DSB damage and activates DNA damage response pathways. (A) The expression of CAS9 in human fibroblasts activates DNA damage responses. The expression of CAS9 was induced with 2 µg/mL Doxy treatment. The relative levels of phosphorylation of BRCA1, CHK1, CHK2 and p53 are indicated at the bottom. n = 3. Data are presented as mean value ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. (B) The expression of CAS9 increased the number of γH2AX foci in human fibroblasts. CTL or CAS9, human fibroblasts with CAS9 inducible expression cassette plated on chamber slides were treated with or without 2 µg/mL doxyc

ycline for 3 days. The expression of CAS9 and γH2AX foci was revealed by immunoflourescence analysis. Representative images are shown. Scale bar, 10 µm. Unpaired t test. n = 20. Data are presented as mean values ± SD. ***P < 0.001. (C) CAS9 induces DNA DSB damage in human fibroblasts. CTL, human fibroblasts with lentiviral empty vector were treated with 2 µg/mL doxycycline for three days; Doxy, Dox, Doxy + Dox, human fibroblasts with lentiviral CAS9 inducible expression vector were treated with 2 µg/mL doxycycline for 3 days or 0.5 µmol/L Dox for 2 h or 2 µg/mL doxycycline for three days + 0.5 µmol/L Dox for 2 h, respectively. Representative images are shown. n = 40. Unpaired t test. Data are presented as mean value ± SD. **P < 0.01