Phospho histone h3 s10

Western blot analysis was performed using standard procedures for whole-cell extracts from cell lines. Antibodies used include Chk2 (1:5000, Becton Dickinson and Millipore), SIRT1 (1:2000, Millipore), acetyl-lysine (1:1000), phospho-CHK2-T68 (1:1000), phospho-CHK2-Thr387 (1:1000), phospho-p53-Ser20 (1:1000), acetyl-p53-Thr382 (1:1000), phospho-histone H2A.X (Ser139) (1:1000), phospho-ATM-Ser1981 (1:1000), ATM (1:1000), phospho-histone H3-S10 (1:1000), p-CDC25C (ser216) (1:1000), CDC25C (1:1000), cleaved PARP-1 (1:1000) and cleaved caspase-3 (1:1000) (Cell Signaling Technology), phospho-CHK2-Thr432 (1:500, Invitrogen), P53 (Do-1, 1:1000, Santa Cruz Biotechnology), FLAG (clone M2) (1:2000), α-tubulin (1:5000, Sigma), and β-actin (1:5000, Sigma). For immunoprecipitation analysis, cell lysates (1–4 mg) after preclearing were mixed with antibodies (2 μg) at 4 °C overnight followed by the addition of 30 μl of protein-G (for mouse antibodies)- or protein-A (for rabbit antibodies)-coupled sepharose beads (GE) for 3 h at 4 °C. Immune complexes were washed three times with lysis buffer [50 mM Tris (pH 7.4), 1% Triton X-100, 0.5% Nonidet P-40, 150 mM NaCl, protease, phosphatase inhibitor mixture (Sigma)]. After boiling in 2× loading buffer, samples were subjected to SDS-PAGE, and then scanned using ECL.

HCT‐116 DNA2^Flox/+/+ (WT) and DNA2^Flox/−/− (inducible deletion) cells were gifts from Dr. Eric Hendrickson at the University of Minnesota. Exon 2 of one copy of the DNA2 gene was flanked by LoxP sites, while the other two copies were either disrupted (DNA2^Flox/−/−) or intact (DNA2^Flox/+/+). Cre recombinase was inducible by 1 μM 4‐hydroxytamoxifen (4‐OHT), which led to excision of DNA2 exon 2. All cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin. 4‐OHT (cat# T176) was from Sigma‐Aldrich. The DNA2 inhibitor C5 was developed in our laboratory (Liu et al, 2016). The ATR inhibitor (VE‐821, cat# A2521) was from ApexBio Technology. Antibodies against phospho‐Chk1 (S345; cat# 2348), Chk1 (cat# 2360), phospho‐histone H3 (S10; cat# 9701), and GAPDH (cat#2118) were from Cell Signaling Technology. The antibody against ATR (cat# sc‐1887) was from Santa Cruz Biotechnology. The DNA2 (cat# ab96488) antibody was from Abcam. The CENP‐A (cat# GTX13939) and phospho‐ATR (T1989; cat# GTX128145) antibodies were from GeneTex.

Immunohistochemistry was performed as previously described [45 (link)]. The following antibodies were used: phospho-STAT3 Y705, phospho-Histone H2AX S139, phospho-Histone H3 S10, and cleaved Caspase 3 (all from Cell Signaling Technology, Danvers, MA). After image acquisition using an Aperio Scanscope XT (Leica), the staining indexes were calculated dividing the number of positive epithelial cells/nuclei by the total number of epithelial cells/nuclei, using Aperio Imagescope software (Leica Biosystems). The resulted ratio is shown as index.