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. 2019 Sep 5;10(4):615–627. doi: 10.1016/j.apsb.2019.08.008

Figure 4.

Figure 4

Induction of reactive oxygen species (ROS) and DNA damage in BRCA1−/− cells by entinostat. (A) The effect of entinostat on cellular ROS in HCC1937 BRCA1 isogenic cells was tested. HCC1937 BRCA1 isogenic cells were treated with 5 μmol/L entinostat for 6 h and intracellular ROS was detected under a flow cytometry with CellROX green fluorescence dye. (B) The effect of entinostat on DNA damage in HCC1937 BRCA1 isogenic cells was tested with the comet assay. HCC1937 BRCA1 isogenic cells were treated with entinostat alone (5 μmol/L) or in combination with N-acetylcysteine (NAC, 5 mmol/L) for 24 h and the DNA damage levels were assessed by comet assay. Scale bar = 100 μm. (C) The DNA tails (50 tails/group) were measured by Image J software and the data were plotted with Graphpad prism 6.0. (D) and (E) The effect of entinostat on cell viability in HCC1937 BRCA1 isogenic cells and its reversal by NAC was tested. Representative cell images (D) and alamarblue data (E) are shown. Scale bar = 400 μm. (F) The effect of entinostat on thioredoxin (TXN) activity was examined. HCC1937 BRCA1 isogenic cells were treated with 5 μmol/L entinostat for 6 h and intracellular TXN activity was measured with a Fluorescent Thioredoxin Activity Assay Kit. (G) The effect of TXNIP silencing on entinostat-induced ROS in HCC1937 BRCA1−/− cells was tested. HCC1937 BRCA1−/− cells were transfected with 2 nmol/L TXNIP siRNA for 24 h prior to dosing with 5 μmol/L entinostat. After 6 h incubation, intracellular ROS was measured with CellROX green kit. (H) The effect of TXNIP silencing on entinostat-induced synthetic lethality in HCC1937 BRCA1−/− cells was tested. HCC1937 BRCA1−/− cells were transfected with 2 nmol/L TXNIP siRNA for 24 h prior to dosing with 5 μmol/L entinostat. After additional 72 h incubation, the cell viability was assessed with the alamarblue assay. *P < 0.05; **P < 0.01 between two indicated groups. (I) The reversal effect of TXNIP silencing on entinostat-induced oxidative stress and DNA damage responses was examined in HCC1937 BRCA1−/− cells. Data are mean ± SD, *P < 0.05, **P < 0.01 between two groups.