Figure 6.

LKB1 deficiency enhances ROS-induced DNA damage. (a and b) Comet assay demonstrating elevated DNA damage in LKB1 deficient cells treated with ROS. U2OS/shR-Ctrl and U2OS/shR-LKB1 cells were treated with 10 μM SB203580 for 2 h followed by the treatment of H₂O₂ (500 μM) for 30 min. The cells were then trypsinized and washed with PBS. Two thousand cells were mixed with 100 μl low melting agarose for alkaline comet assay. Cells in the gel were stained and visualized with epifluorescence microscopy (a). (b) Percentage of DNAs in the tail (damaged DNA) was calculated. *P < 0.01; N.S., not significant (n = 3). (c) DNA damage escalates in LKB1 deficient cells. U2OS/shR-Ctrl and U2OS/shR-LKB1 cells were treated with different dosages of H₂O₂ for 1 h. WCEs were isolated and analyzed with Western blot. GAPDH served as loading control. (d) NAC reduces LKB1-deficiency-induced DNA damage. U2OS/shR-Ctrl and U2OS/shR-LKB1 cells were pretreated with 10 μM SB203580 or 5 mM NAC for 2 h, followed by treatment with H₂O₂ (200 μM) for 1 h. After the treatment, WCEs were isolated for Western blot analysis. GAPDH served as loading control. (e) p38-CA alleviates LKB1-deficiency-induced DNA damage. U2OS/shR-Ctrl and U2OS/shR-LKB1 cells with stable expression of p38-CA were treated with 200 μM H₂O₂ for 1 h. After the treatment, WCEs were isolated for Western blot analysis. GAPDH served as loading control.