Phospho histone h2a x ser139 d7t2v

Bulk arsenic was purchased from Sigma-Aldrich and stored in the dark with argon protection. Human normal liver cells (HL-7702), human embryonic kidney 293 (HEK293), human non-small cell lung cancer (A549), and human breast cancer cell (MCF-7) were obtained from ATCC. DPBF, MB, H₂O₂ (30%), glutathione, and N-methyl-pyrrolidone (NMP) were purchased from Sigma-Aldrich. RPMI 1640 medium, Dulbecco’s modified Eagle medium (DMEM), PBS, fetal bovine serum (FBS) and trypsin-EDTA were purchased from Gibco Life Technologies. Primary antibodies: Phospho-Histone H2A.X (Ser139) (D7T2V), Cell Signaling (Product # 80312), Dilution 1:200; C-CAS3 (Asp175) (5A1E), Cell Signaling (Product # 9664), Dilution 1:250. Secondary antibodies: Anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488, ThermoFisher (Catalog # A-11034), Dilution 1:1000; Anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647, ThermoFisher (Catalog # A-21236), Dilution 1:1000.

Cells were washed 2 × with 1 × PBS and counted. Then, 5 × 10⁴ cells in 5–10 µL were seeded per reaction field onto adhesion slides (Marienfeld #0900000). After a pre-extraction step with 0.1% Tween in PBS (PBST), cells were fixed with 2% PFA in PBS for 20 min at RT. Cells were permeabilized with 0.5% Triton in PBS for 10 min at RT. Slides were washed 3 × with PBS. Cells were blocked with 5% BSA in PBST for 1 h at RT. Primary antibodies were added and slides were incubated o/n at 4 °C. The next day, slides were washed 3× with 5% BSA in PBS and incubated with secondary antibodies for 1 h at RT. Slides were washed 3 × with 5% BSA in PBS and 1 × with PBS. DAPI solution was added (0.2 μg/mL DAPI in PBS) and incubated for 10 min at RT. Slides were washed 2× with PBS and coverslips were mounted with DAKO mounting medium (Agilent Technologies, Santa Clara, CA, USA). Slides were dried o/n at 4 °C before imaging. Slides were imaged using an LSM 700 laser scanning confocal microscope (Carl Zeiss). Primary antibodies used: 1:1000 γH2A.X (Phospho-Histone H2A.X Ser139 D7T2V, Cell Signaling #80312S) in blocking solution, 1:200 TP53BP1 (Bethyl #A300-272A) in blocking solution. Secondary antibodies used: 1:2000 Alexa Fluor 546 goat anti mouse (Invitrogen #A-11018) and 1:2000 Alexa Fluor 488 goat anti rabbit (Invitrogen #A-11070) in blocking solution.

Phospho-Histone H2AX (Ser139) (D7T2V), Cell Signaling (Product # 80312), Dilution 1:200; Cleaved Caspase-3 (Asp175) (5A1E), Cell Signaling (Product # 9664), Dilution 1:250; Rat anti mouse CD11c, Miltenyi Biotec (Product 130-128-247), clone number: REA618, Dilution 1:200; Rat anti mouse CD86, Miltenyi Biotec (Product 130-102-558), clone number: PO3.3, Dilution 1:100; Rat anti mouse CD80, Miltenyi Biotec (Product 130-117-683), clone number: 2D10, Dilution 1:100.