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. 2010 Nov 17;5(11):e14027. doi: 10.1371/journal.pone.0014027

Figure 4. The DNA damage-activated PIKKs family members ATM, ATR and DNA-PK are not required for downregulation of BRCA1.

Figure 4

A) Immunoblotting detection of BRCA1 in HeLa cells treated with 10 Gy IR or 200 µM MMS. B) BRCA1 downregulation is not blocked by caffeine. Immunoblotting detection of BRCA1 in HeLa cells pre-treated with 10 mM caffeine for 30 min prior to 200 µM MMS treatment for 6 hrs. C) The downregulation of BRCA1 is not prevented by the ATM inhibitor (KU-55933). Immunoblotting detection of BRCA1 in HeLa cells pre-treated with 10 µM KU-55933 for 30 min prior to 200 µM MMS treatment for 6 hrs. γH2AX and pChk2 detection were used as controls to confirm inhibition of ATM and/or ATR kinases. D) BRCA1 is downregulated in ATM-deficient human fibroblasts. Cells were treated with 200 µM MMS treatment for 6 hrs and harvested for immunoblotting. E) Depletion of ATR by RNAi does not prevent BRCA1 downregulation by MMS. Left, immunodetection of ATR following shRNA constructs transfection and puromycin selection. Right, ATR-depleted cells were treated with 200 µM MMS and harvested at the indicated times for immunoblotting. F) BRCA1 is downregulated in DNA-PKcs deficient cells. Glioblastoma DNA-PKcs proficient (MO59K) or deficient (MO59J) were treated with 200 µM MMS and harvested at the indicated times for immunoblotting.