Figure 8. ATR dependence of non-thermal plasma induced phosphorylation of H2AX.

(A) Immunoblot of γ-H2AX (top), and α-tubulin (bottom) from MCF10As exposed to non-thermal plasma at a dose of 1.95 J/cm² or 200 µM H₂O₂ in the presence (+) or absence (−) of 100 µmol/L Wortmannin (Wort.) or 10 µmol/L KU55933 (KU). (B) MCF10As were depleted of endogenous ATM by shRNA for 72 hours (bottom, immunoblot of ATM after ATM or non-targeting (NT) shRNA). Cells were then plated on glass cover slips and exposed to DBD plasma at a dose of 1.95 J/cm² or 200 µM H₂O₂. (C) MCF10As depleted of endogenous ATR by shRNA for 72 hours (bottom, immunoblot of ATR after ATR or non-targeting (NT) shRNA). (B,C) After knockdown, cells were plated on glass cover slips for 24 h followed by exposure to non-thermal plasma at a dose of 1.95 J/cm² or 200 µM H₂O₂. After one-hour incubation, lysates were prepared and resolved by SDS-PAGE and representative immunoblots with antibody to γ-H2AX (top) or α-tubulin (bottom) are shown.