Priority Research Paper Volume 12, Issue 6 pp 4688—4710

ATM is a key driver of NF-κB-dependent DNA-damage-induced senescence, stem cell dysfunction and aging

Figure 1. DDR and NF-κB are activated concomitantly in senescent MEFs and aged tissues. (A) Immunoblot detection of p-p65 and total p65 in liver tissue from 16-week-old WT (n=3) and Ercc1-/Δ (n=3) mice. (B) Immunoblot detection of phosphorylation of ATM and downstream targets γH2AX and p21 in liver from 16-week-old WT and Ercc1-/Δ mice. (C) Immunoblot detection of phosphorylation of NF-κB and IκBα in liver lysates from 3, 12 and 24 month-old WT mice. n=3 mice per group. (D) Immunoblot detection of p-ATM, ATM and p21 in the same liver lysates. (E) Immunoblot detection of DDR effectors in nuclear extracts from passage 5 WT and Ercc1-/- MEFs, grown at 20% oxygen. (F) Level of NF-κB activation is higher in Ercc1-/- MEFs compared to WT MEFs at passage 5, as measured by Immunoblot detection of p-p65 and total p65 in WT and Ercc1-/- MEFs at passage 5 after culturing in 20% oxygen. (G) Representative images of immunofluorescent detection of p65 and NEMO in passage 4 WT and Ercc1-/- MEFs grown at 20% oxygen. Blue: DAPI staining; Green: p65 (top panel) or NEMO (bottom panel). Images were taken at the magnification of 60x.