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. 2015 Sep 28;19(12):2851–2864. doi: 10.1111/jcmm.12678

Figure 5.

Figure 5

ROS‐dependent DNA damage in cFP‐treated INT‐407 cells (A) The level of ROS is elevated by 1 mM cFP treatment in INT‐407 cells. However, 1 mM linear FP dipeptides (FP) treatment does not elevate level of ROS. After cells were incubated with 1 mM cFP in conjunction with 0.5 mM N‐acetylcysteine (NAC) or H2O for 48 hrs, ROS level was determined by flow cytometry using DCFDA. Untreated cells (CTR) were used as a negative control. (B) ROS scavenger treatment decreases cFP‐induced phosphorylation of CHK2 (T68) and ATM (S1981). The lysates were immunoblotted with anti‐phospho CHK2 (T68) and anti‐phospho ATM (S1981) antibodies respectively. As controls, INT‐407 cells were untreated (CTR) or treated with 1 μM doxorubicin (Doxo). Western blots were analysed quantitatively. (C) Representative images demonstrate that ROS scavenger treatment decreases cFP‐induced γH2AX foci formation. After cells were incubated with 1 mM cFP in conjunction with 0.5 mM NAC or H2O for 48 hrs, cells were immunostained with anti‐ phospho H2AX (S139) antibody. Untreated cells (CTR) were used as a negative control; scale bar, 10 μm. (D) ROS scavenger treatment attenuates cFP‐induced DNA damage. After cells were incubated with 1 mM cFP in conjunction with 0.5 mM NAC or H2O for 48 hrs, a neutral comet assay was performed. Untreated cells (CTR) were used as a negative control. (E) Increased cytochrome C release in cFP‐treated INT‐407 cells. The cytoplasmic fraction from 1 mM cFP or linear FP dipeptides‐treated INT‐407 cells was immunoblotted with an anti‐cytochrome C antibody as described in Materials and Methods section. Untreated cells (CTR) were used as a negative control. Western blots were analysed quantitatively. (F) Disturbance of mitochondrial membrane is induced in cFP‐treated INT‐407 cells. After INT‐407 cells were treated with 1 mM cFP or linear FP dipeptides for 48 hrs, cells were stained with 5 μM Rhodamine 123 and flow cytometry was performed. Untreated cells (CTR) were used as a negative control. *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.005.