Phospho histone h3 phh3 ser 10

Protein assays (Pierce, Rockford, IL) and Western blot analysis (Bio-Rad, Hercules, CA) were performed as previously described (Cantara et al., 2012 (link)) with 8 μg of protein loaded/well and protein bands quantified using ImageJ (version 1.46r) with normalization to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Levels of each phosphorylated protein were additionally normalized to total protein levels. Antibodies, their phosphorylated epitope (when relevant), their dilution from stock solution, and their source are as follows: GAPDH (1:10,000; Fitzgerald, Acton, MA), p25/TPPP (clone EPR 3316, 1:1000; Epitomics), phospho–LIMK 1 (Thr-508; 1:500; Abcam), phospho–LIMK 2 (T505; 1:500; Abcam), phospho-cofilin (Ser-3; 77G2, 1:1000; Cell Signaling), cofilin (1:2000; Sigma-Aldrich), phospho-FAK (Tyr-576/577; 1:1000; Cell Signaling), phospho-FAK (1:1000; Tyr-397; Cell Signaling), FAK (1:1000; Cell Signaling), phospho-paxillin (Tyr-118; 1:1000; Cell Signaling), paxillin (1:500; Cell Signaling), phospho–histone H3 (pHH3; Ser-10; 1:1000; Cell Signaling), and histone H3 (1:1000; Cell Signaling).

Cells were seeded in triplicate and then incubated in 0.2 μM WEE1i (AZD1775), 1 μM ATRi (AZD6738) or combination treatments for 8 h or 24 h. Cells were then trypsinized, fixed washed and incubated with blocking buffer. Cells were then stained with the following primary antibodies diluted in blocking buffer at 1:300: γH2AX (catalog 9718, Cell Signaling Technology, Inc), pRPA32 (S33, catalog A300-246A, Bethyl Laboratories, Inc.) or phospho-histone H3 (pHH3, Ser10, catalog 53348, Cell Signaling Technology, Inc). The cells were washed, and incubated with secondary antibody goat anti-Rabbit IgG (H+L), Alexa Fluor® 647 (ThermoFisher Scientific) for 30 min. The cells were then incubated with 50 μg/mL propidium iodide (Sigma-Aldrich) and subjected to flow cytometry acquisition on BD LSRII (BD Biosciences) and data analysis with FlowJo (Tree Star, Inc., Ashland, OR). For Phospho-Histone H3 (Ser10), cells were treated with 0.2 μM WEE1i, 1 μM ATRi or combination for 6 h or 12 h then 500 nM nocodazole for 6 h to prevent cells from exiting mitosis. The supernatant and attached cells were collected and fixed. The staining and detection process was performed same as above.