α γh2ax

Chemicals and cytokines used were staurosporine, cycloheximide, doxorubicin, DAPI, H₂O₂, Nutlin-3, polybrene, propidium-iodide, doxocycline, puromycin (all Sigma), AnnexinV–APC, AnnexinV-488, 7-AAD (Biolegend), etoposide, camptothecin (Alexis). Antibodies: α-IκBα (#9242), α-PARP1 (#9532), α-GAPDH (#2118), α-Histone H3 (Ser10) (#9701), α-γH2A.X (#2577), pChk1 Ser345 (#2341), Chk1 (#2345) (Cell Signaling); α-p53 (sc-6243), α-tubulin (sc-32293) (Santa Cruz Biotechnology); α-PDCD5 (Proteintech); α-LaminB1 (gift from Peter Gruber); α-Flag (M2) (Sigma), α-p21 (#554262) (BD Pharmingen); α-rabbit AlexaFluor 488 (Invitrogen), goat α-rabbit HRP, goat α-mouse HRP (Dako). SiRNA targeting human PDCD5 (5′-CUAAAGCAGUAGAGAAUUATT-3′) was synthesised by Microsynth and transfected into 293T cells using Metafectene (Biontex) according to the manufacturer′s instructions.

Tissue samples were incubated with paraformaldehyde (4%) for 10 min and then washed three times with PBS containing Tween‐20 (0.05%, PBS‐T). The slides were incubated with blocking solution (1% BSA and 5%, goat serum in PBS‐T) for 1 hr at room temperature (or overnight at 4°C) and then washed once with PBS‐T. Slides were then incubated with both α‐cytokeratin‐8/18 (1/10, rat monoclonal, University of Iowa, US) and α‐γH2AX antibody (1/500, 20E3, rabbit monoclonal, Cell Signaling Technologies) for 1 hr (or overnight) at 4°C. After washing with PBS‐T, slides were incubated with both α‐rabbit (Alexa Fluor 546) and α‐rat (Alexa Fluor 488) secondary antibodies (Molecular probe, US). γH2AX and cytokeratin‐8/18 signals were detected using BZ‐9000 (KEYENCE) and LEICA SP8.
LNCaP cell was fixed with ice‐cold methanol for 20 min on ice and permeabilized with 0.5% Triton X‐100 in PBS (0.05% PBS‐T) on ice. After incubation in blocking solution (5%, BSA), cells were incubated with the following antibodies for 1 hr: α‐γH2AX (1/1000, JBW301, mouse monoclonal, Millipore), α‐γH2AX (1/500, 20E3, rabbit monoclonal, CST, US) and α‐Cyclin A (1/500, rabbit polyclonal, ab87359).

Cells were plated at low density in 24-well format containing a Matrigel® (Corning) treated dish. When desired confluence was reached, cells were washed with PBS and fixed with paraformaldehyde (Sigma-Aldrich) at 4% in PBS. The samples were blocked and permeabilised with 5% of BSA (Sigma-Aldrich) (w/v) in PBS and 0.3% of triton x-100 (Sigma-Aldrich) (v/v) for 5 min at room temperature. The washes were performed using PBS with 0.1% of BSA. The cell line CHRF-288-11 was fixed in suspension with paraformaldehyde at 2% in PBS for 20 min and permeabilized with PBS and 0.5% of triton x-100 for 5 min. The cells were dried with PBS over poly-L-lysine coated slides. The staining was performed overnight using a 5% BSA/PBS solution and the desired antibodies. To detect KDM5A and NUP98-KDM5A the same antibody was used, the α-KDM5A (ab70892, Abcam). Also used α-γH2AX (#9718, Cell Signaling Technology, Danvers, MA, USA), α-tubulin (sc-23948, Santa Cruz; Dallas, TX, USA), α-CDC20 (sc-13162, Santa Cruz) and α-MRNP41 (sc-374261, Santa Cruz). For mitochondria staining, Mitotracker 633 reagent (Invitrogen) was used at 200 nM for 40 min following manufacturer instructions. Then the dish was mounted and stained for DNA (DAPI) using VECTASHIELD® Antifade Mounting Media (Vector Laboratories, Burlingame, CA, USA).