Fig. 3

Deletion of CgCKB1 or CgCKB2 increases cellular apoptosis and DNA breaks. (A) Cells were treated with 0.01% MMS or 4 µM 4-NQO or left untreated for 2 h and stained with Annexin V-FITC and PI. The Fluorescence intensity was detected by BD Fortessa Flow cytometer. Q1 (Annexin V-/PI+): dead cells; Q2 (Annexin V+/PI+): necrotic cells; Q3 (Annexin V+/PI-): apoptotic cells; Q4 (Annexin V-/PI-): live cells. (B) The percentages of each strain that are apoptotic (black bars) and death (gray bars) were obtained. The relative cell ratio was compared with WT in RPMI 1640 medium (set at 1.0) (C) Representative fluorescence micrographs showing DNA fragmentation and unusual chromosome agglutination. Cells were incubated in RPMI 1640, 0.01% MMS or 4 µM 4-NQO for 12 h at 30 ℃ and then stained with DAPI and FITC. The DNA fragmentation was visualized with FITC (green fluorescence) and unusual chromosome agglutination was visualized with DAPI (blue fluorescence). Data were representative of three independent experiments. (D) The TUNEL-positive cells were quantified. DNase I treatment was used as the positive control. The percentages of TUNEL-positive cells were presented as mean ± SD and assessed for statistical analysis by one-way ANOVA followed by Tukey test; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.