γh2ax

Immunocytochemical staining was used to visualize the DSBs and distinguish between the cell cycle stages G1 and S/G2. Four hours before fixation, a nucleoside analogue of thymidine, 5-ethynyl-2′-deoxyuridine (EdU; Thermo Fisher Scientific), was added at a final concentration of 10 μM. The cells were fixed in 4% paraformaldehyde and permeabilized in 0.2% Triton-X (both from Sigma-Aldrich, St. Louis, MO, USA). Overnight incubation with primary antibodies against γH2AX (Biolegend, San Diego, CA, USA) and 53BP1 (Santa Cruz Biotechnology, Dallas, TX, USA) was followed by 1 h of incubation with a secondary antibody conjugated with Alexa 555 (Cell Signaling Technology, Danvers, MA, USA). The samples were stained with the Click-iT® EdU Alexa Fluor® 488 Imaging Kit (Thermo Fisher Scientific) to visualize EdU according to the manufacturer’s instructions. Finally, the nuclei were stained with Hoechst dye (BisBenzemide H33342; 1 μg/ml; Sigma-Aldrich).

Cells (2 × 10⁴) were transfected with siRNA, seeded on glass slides (Merck Millipore, Burlingame, CA, USA), cultured in DMEM supplemented with 10% FBS for 24 h, and irradiated with 10 Gy. After 0.5 h (for γ-H2AX and pDNA-PKcs) or 24 h (for γ-H2AX), cells were fixed for 30 min with 4% paraformaldehyde in PBS. Then, cells were washed with PBS-T for 5 min and blocked with PBS-T containing 5% BSA for 5 min on ice. Next, cells were incubated for 2 h at room temperature with primary antibodies against phosphorylated histone H2AX (γ-H2AX; 1:400; cat# 613401; BioLegend, San Diego, CA, USA), IGFBP-3 (1:50; H-98; Santa Cruz Biotechnology, Dallas, TX, USA), and S2056-phopho-DNA-PKcs (1:200; ab18192; Abcam, Cambridge, UK) in PBS-T with 1% BSA. glass slides were then washed with PBS-T three times (5 min each), and cells were incubated at room temperature for 1.5 h with secondary antibodies (Alexa Fluor 488 and 594 donkey anti-rabbit and/or anti-mouse IgG; Life Technologies) in PBS-T with 1% BSA. After washing with PBS, cells were counterstained and mounted with Vectashield (Vector Laboratories, Inc., Burlingame, CA, USA) and then imaged using a fluorescent microscope (BZ-X700; Keyence, Osaka, Japan). Cells were considered γ-H2AX-positive if they had >10 foci per nucleus [41 (link)].

We used commercially available antibodies for the following antigens: Aβ (1:500, clone 6E10, BioLegend), γH2Ax (1:1000, #2577), H2Ax (1:1000, #7631), Histone H3 (1:1000, #9717), Caspase3 (1:1000, #9662), cleaved Caspase-3 (1:1000, #9661), α-Tubulin (1:1000, #2144, Cell Signaling Technology, MA), NeuN (1:500, EPR12763, abcam, UK), MAP2 (1:1000, clone Ap20, BD Biosciences, NJ), AT8 (1:1000, MN1020), AT180 (1:1000, MN1040), AT100 (1:1000, MN1060), ZO-1 (1:500, 40-2200), Tau5 (1:1000, AHB0042, Thermo Fisher Scientific, MA), γH2Ax (1:1000, host mouse, clone JBW301,#05-636), Tau-1 (1:1000, clone PC1C6, MAB3420), Olig2 (1:500, AB9610), β-actin (1:10000, A5441), H3K9me3 (1:1000, 05-499), Tau oligomeric (T22, 1:1000,#ABN454, millipore, CA), LaminB (1:1000, M-20, sc-6217), β Tubulin (1:1000, sc-5274), GAPDH (1:5000, FL-335, sc-25778, santa cruz biotech.), GFAP (1:500, G 3893, Merck, DE), mouse tau (1:1000, 012-26963), and Iba1 (1:500, 013-27691, FUJIFILM Wako Chemical Coporation, JP); mouse, rabbit and goat IgG HRP-conjugated (Jackson ImmunoResearch, PA).