Fig. 2

Effects of deleting the 13q-Enh on Beas-2B cells. a Soft agar assays for the wild-type and 13q-Enh^−/− Beas-2B cells (C5 and C23) treated by NNK for 15 generations. The corresponding colony number is indicated at the lower left corner of each picture. b Knockout of the 13q-Enh promoted NNK-induced malignant transformation of Beas-2B cells. The colony number ratios of NNK over DMSO for wild-type and 13q-Enh^−/− clones were compared to correct the possible change of “baseline” colony formation caused by knockout of this enhancer. (n = 3 per group; error bars are SD; **p < 0.01, *p < 0.05, unpaired Student’s t test). c γ-H2AX analysis of the cells treated with NNK for 15 generations to evaluate DNA damages in the wild-type cells and 13q-Enh^−/− clones. The nuclei were stained with Hoechst 33258 (blue), and breaking DNA ends were detected with γ-H2AX antibody (red). d The γ-H2AX signal ratios of NNK over DMSO for the wild-type and 13q-Enh^−/− clones were compared. (n = 3 per group; error bars are SD; **p < 0.01, *p < 0.05, unpaired Student’s t test). e DNA repair efficiency of the wild-type Beas-2B cells and the two 13q-Enh^−/− clones were compared using host-cell reactivation (HCR) as described in the “Materials and methods” section. DNA repair efficiency was reflected by luciferase activity. The results indicated that deletion of the enhancer significantly impaired the DNA repair efficiency. (n = 3 per group; error bars are SD; **p < 0.01, unpaired Student’s t test). f Cell apoptosis analysis of the H₂O₂-treated wild-type Beas-2B and 13q-Enh^−/− clones. Cells treated with 100 μM H₂O₂ were incubated for 4 h and harvested for Annexin-V/d PI double staining followed by flow cytometry analysis. g Statistical analysis of cell apoptosis percentages. (n = 3 per group; error bars are SD; **p < 0.01, *p < 0.05, unpaired Student’s t test)