The roles of BRCA1, CHK1, and RAD51 in cytarabine- or DNR-induced DNA DSBs and apoptosis in THP-1 cells.

THP-1 cells were infected with BRCA1, CHK1, RAD51, or non-target control (NTC) shRNA lentivirus overnight, washed and then treated with 4 µM cytarabine or 25 nM DNR for 48 h. shRNA knockdown of BRCA1, CHK1, or RAD51, and induction of γH2AX by cytarabine or DNR were determined by Western blotting (Panels A–C). The lane headings indicate the treatment conditions ‘Control’ ‘Ara-C’ or ‘DNR’ and the+or – indicate the shRNA-treated cells from which the sample was derived. Apoptotic events in the cells were determined by annexin V/PI staining and flow cytometry analyses (Panel D). THP-1 cells were infected with BRCA1, CHK1, or RAD51 shRNA lentivirus overnight. The cells were washed three times with complete medium and cultured in virus-free complete medium for up to 72 h. The cells were then treated with 50 µM cytarabine or 2 µM DNR for 3 h and the drugs were washed out, and the cells were cultured in drug-free complete medium for up to 8 h. DNA damage was assessed by COMET assay. Representative images at the 8 h time point are shown (Panel E). The median percent DNA in the tail from at least three replicate gels are shown plus or minus the standard error of the mean (Panel F). *indicates p<0.05.