Primary antibody against γ h2ax

Forty-eight hours following transfection with miR-101 mimic or miRNA mimic negative control, 1×10⁵ cells were seeded in chamber slides and incubated overnight. The cells were subsequently exposed to 6 Gy irradiation (IR). Twenty-four hours following IR, the cells were fixed in 4% paraformaldehyde (Sigma-Aldrich), permeabilized in 0.1% Triton X-100 (Sigma), blocked in 2% bovine serum albumin (Roche, Stockholm, Sweden) and incubated with a primary antibody against γ-H2AX (Abcam, San Francisco, CA, USA) overnight at 4°C. The primary antibody was subsequently washed off, and a secondary antibody conjugated to fluorescein isothiocyanate (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was applied to the slides. Cells were washed with phosphate-buffered saline (Sigma-Aldrich) and counterstained with DAPI (Invitrogen Life Technologies). The γ-H2AX foci were observed under a fluorescence microscope (Olympus BX51, Olympus). For each group, the γ-H2AX foci were counted in ≥50 cells.

NSCLC cells were seeded on 12-well plates with coverslips, fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, blocked with 1% BSA, and incubated with the primary antibody against γ-H2AX (diluted 1:200; Abcam, UK) overnight at 4°C. After washing, cells were incubated with the fluorescent secondary antibody, and coverslips were mounted using fluorescence quenching mounting medium with DAPI. Fluorescence images were captured using a microscope.

DNA damage in PC-9 cells, H3255 cells, or JAK1 knockdown PC-9 cells was determined using immunofluorescence with γ-H2AX. After different treatments, cells were fixed using 4% paraformaldehyde, permeabilized, and incubated with 5% goat serum dissolved in 0.2% Triton X-100 PBS buffer for blocking. Then, the samples were incubated with the primary antibody against γ-H2AX (1:200, Abcam, Cambridge, UK), followed by incubation with the fluorescently labeled secondary antibody (Aviva Systems Biology, California, USA), and nuclei were counterstained with Vectashield DAPI mounting medium (Vector Labs, CA, USA). Finally, images were acquired using confocal microscopy (Keyence, Tokyo, Japan).