Ti a1 capture system

Cells seeded onto coverslips in 6-well plates (2.8 × 10⁵ cells per well) were treated with miR-1587 mimics or miR-NC. After 48 hours, cells were fixed in 4% paraformaldehyde, permeabilized in 0.3% Triton X-100 in PBS, and blocked in 3% bovine serum albumin in PBS. Subsequently, cells were immunostained with anti-γ-H2AX (1:400; Millipore) primary antibodies overnight at 4 °C in a humidified environment. Cells were washed and labeled with FITC-conjugated anti-IgG antibodies (1:400; KPL) at room temperature for 2 hours. Nuclei were visualized with 4, 6-diamidino-2-phenylindole–staining (2 µg/mL) for 10 minutes in the dark. Images were acquired using a Nikon Ti-A1 capture system (magnification, 60×). The number of γ-H2AX foci per nucleus was counted.

Cells were cultured on coverslips in six-well plates (2.5 × 10⁵ cells/well). At 24 h or 48 h post-transfection, HeLa and U2OS cells were fixed in 4% formaldehyde for 15 min at room temperature, permeabilized in 0.3% Triton X-100-PBS buffer and then blocked in 3% bovine serum albumin for 1 h at room temperature. Cells were stained using standard procedures. Specifically, cells were incubated with phospho-histone H2AX (Ser139) monoclonal antibody (Millipore, USA; Cat. No. 05-636, 1:500) at 4°C overnight in a humidifier, and then washed twice in PBS. Subsequently, cells were incubated with a FITC-labeled anti-IgG antibody (KPL, USA; Cat. No. 02-15-06, 1:250) at room temperature for 2 h. DNA was stained with DAPI at a concentration of 2 μg/ml for 10 min in the dark. Images were obtained using a Nikon Ti-A1 capture system (magnification, 60×).