Fig 2: LNCaP cells were exposed to 10 µM ENZ for 24 hrs prior to, during irradiation (2 Gy) and for up to 24 hrs after irradiation.At the indicated times cells were processed for immunoblotting. A) LNCaP nuclear lysates immunoblotted for pS1981 ATM and TBP (TATA-binding protein). No XRT represents sham treatment. B) Ratio of pS1981 ATM immunoreactive protein corrected for TBP immunoreactive protein.

Fig 3: Scheme depicting role of HORMAD1 in DSB signaling and repair. Our immunofluorescence microscopy analyses (Fig. 4) show that formation of CtIP, RAD51 and RPA p34-containing IRIF is reduced in HORMAD1-depleted cancer cells (whereas 53BP1 and p95/NBS1 foci remain intact in the absence of HORMAD1). We infer that HORMAD1 contributes to HR pathway activation at (or prior to) the CtIP-mediated resection step, but does not affect the proximal stages of DSB signaling. HORMAD1-depletion leads to attenuation of DR-GFP/HR reporter activity (Fig. 3), prolonged IR-induced ATM signaling (Fig. 4), and radiosensitivity (Fig. 3), further demonstrating that HORMAD1 promotes the HR pathway.

Fig 4: Pharmacologic inhibition of ATM rescues oxidative stress-induced senescence by suppressing ATM- and NEMO-mediated NF-?B activation. (A) Representative images of primary WT and Ercc1-/- MEFs were induced to undergo senescence by serial passaging at 20% oxygen. At passage 5, MEFs were grown in the presence or absence of KU-55933 (10 µM) for 72 hrs. Senescence was determined by SA-ßgal staining. Images were obtained at the magnification of 10x. (B) Quantitation of the percent SA-ßgal positive cells. Graph represents the mean +/- s.e.m. of three independent experiments. Student’s t-test, ***p <0.001, ****p <0.0001. (C) Passage 5 Ercc1-/- MEFs treated with vehicle or KU-55933 (10 µM) for 72 hours were collected and levels of p21 and p16INK4a were determined by western blotting. (D) Passage 5 Ercc1-/- MEFs were treated with KU-55933 (10 µM) for 72 hours and whole cell lysate (CL) and nuclear extracts (NE) were analyzed by immunoblotting for expression of proteins involved in the DNA damage response. (E) Whole cell lysate (CL) and nuclear extract (NE) were extracted from Ercc1-/- MEFs treated with 10 µM of KU-55933 for analysis of nuclear NEMO and p65. GAPDH was used as a loading control of total proteins and LaminA/C as a loading control of nuclear protein. (F) Passage 5 WT and Ercc1-/- MEFs transfected with a NF-?B-luciferase reporter construct were cultured in the presence or absence of KU-55933 (10 µM) and were collected for luciferase assays after 72 hours. (G) qRT-PCR analysis of mRNA expression in passage 5 WT and Ercc1-/- MEFs treated with or without of KU-55933 (10 µM) for 72 hrs. P values were determined using a Student’s t-test. *p<0.05, **p<0.01, ***p <0.001.

Fig 5: Depletion of PCNA or Pol? recapitulates the H2O2-induced ATM hyper phosphorylation phenotype of Rad18-depleted HDF. (A) Replicate cultures of HDF were transfected with siRNA against PCNA or non-targeting control siRNA, synchronized in G1 and treated with H2O2.One hour later, cell extracts were isolated and analysed by SDS–PAGE and immunoblotting with the indicated antibodies. Bands corresponding to ATM (S1981-P) were quantified by densitometry. The amount of ATM (S1981-P) in each lane is expressed relative to the amount of ATM (S1981-P) in scrambled control siRNA-transfected HDF cells that were treated with H2O2. (B) Replicate cultures of HDF were transfected with siRNA against Pol? or non-targeting control siRNA, synchronized in G1 or S-phase and treated with H2O2, UVC or ultraviolet A. After 1 h, cell extracts were isolated and analysed by SDS–PAGE and immunoblotting with the indicated antibodies. (C) Control or YFP–Pol?-overexpressing cells were treated with H2O2 or UVC. One hour later, cell extracts were isolated and analysed by SDS–PAGE and immunoblotting with the indicated antibodies. The amount of ATM (S1981-P) in each lane is expressed relative to the amount of ATM (S1981-P) in control H2O2-treated cells.