Lmagarose

The comet assay was performed using a reagent kit for single‐cell gel electrophoresis (Trevigen, Inc., Gathersburg, MD, USA) according to a modified method of Viera et al. [14 (link)]. Cells were trypsinized and collected, washed with PBS, and resuspended on PBS. Cells were added to melted LMAgarose (Trevigen) cooled to 37 °C at a ratio of 1 : 10 and pipetted onto a pre‐warmed comet slide and spread evenly. Slides were then placed at 4 °C for 30 min to allow adherence of the agarose to the slides. The slides were then gently immersed in lysis solution overnight at 4 °C. Following lysis, the slides were immersed in 1× Neutral Electrophoresis Buffer containing Tris base and sodium acetate (corrected to pH 9 with glacial acetic acid) for 30 min at 4 °C. The slides were electrophoresed at 21 V for 45 min at 4 °C in the neutral electrophoresis buffer and then immersed in DNA precipitation solution and 70% ethanol successively for 30 min each time. Then, slides were stained with DAPI and viewed using fluorescent microscopes. Comet measurement and quantitative analysis were performed using casp software.

The formation of DNA double-strand breaks (DSBs) were assessed by the alkaline version of single-cell electrophoresis (Comet Assay). The cell suspension was mixed with LMAgarose (Trevigen, Gaithersburg, MD, USA) in a ratio of 1 to 10 (30 to 300 μL, respectively) and incubated in a lysis solution overnight. The slides were further placed into an alkaline buffer solution (200 mM NaOH, 1 mM EDTA (pH > 13)) for 1 h and followed by a horizontal electrophoresis for 40 min, 25 V, and 300 mA. Next, the slides were washed twice for 1 min with deionized water and fixed in 70% ethanol for 5 min. SYBR Green I (Trevigen, Gaithersburg, MD, USA) (10,000×) was diluted with a TE buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA). Finally, the slides were examined by fluorescent microscopy (Olympus BX63, Tokyo, Japan). The tail moment (TM) and olive tail moment (OTM)) were calculated using the ImageJ software. At least 50 comets were analyzed for each experiment. Differences between the control and treated cells in the tail moment (TM) and olive tail moment (OTM) were analyzed by the Kruskal-Wallis test followed by Dunn’s test with the Benjamini-Hochberg adjustment in R software (R Foundation for Statistical Computing, Vienna, Austria; URL https://www.R-project.org/).

Opti-MEM reduced serum medium and Lipofectamine 2000 reagent were purchased from Invitrogen (Carlsbad, CA, USA). Protease inhibitor cocktail tablets (Complete Mini EDTA-free) were purchased from Roche (Mannheim, Germany). Primary antibodies utilized were poly-clonal rabbit anti-human TRPM2 antibody (cat. #A300-414A; Bethyl Laboratories, Montgomery, TX, USA), polyclonal rabbit anti-human β-actin (cat. #600-401-886), polyclonal rabbit anti-human apoptosis-inducing factor (AIF) (cat. #200-401-985) (both from Rockland Immunochemicals, Limerick, PA, USA) and monoclonal mouse anti-human poly(ADP-ribose) glycohydrolase (PARG) clone D8B10 (cat. #MABS61; Millipore). Two secondary antibodies, horseradish peroxidase (HRP)-conjugated goat anti-rabbit and HRP-conjugated rabbit anti-mouse were purchased from Jackson ImmunoResearch (West Grove, PA, USA). The CometAssay kit, which includes alkaline lysis solution, LMAgarose, 2-well CometSlides and SYBR-Green was purchased from Trevigen (Gaithersburg, MD, USA).

Non-tumorigenic murine alveolar type II epithelial cells (C10) were challenged with hyperoxia and/or radiation and processed for comet assay analysis as per manufacturer’s instructions (Trevigen, Gaithersburg, MD, USA). Briefly, cells (1 × 10⁵ cells/mL in PBS) were mixed with LMAgarose^® (1:10, v/v) and immediately pipetted onto Trevigen’s CometSlides™. Cells were then lysed (4 °C, 30 min) and kept in dark for unwinding at room temperature. Electrophoresis was done in a horizontal electrophoresis unit at 18 volts (300 mA) for 25 min. Slides were washed twice with deionized water, fixed in 70% ethanol and dried at 45 °C. DNA was stained by SYBR green (Trevigen). At least 150 cells were scored per group. Visual analysis of cells and comet tail length was measured using Comet Image Analysis software (Comet Assay IV, Perceptive Instruments Ltd., Haverhill, UK). Images were captured on an Olympus IX51 fluorescence microscope using a monochrome CCD FireWire camera (Perceptive Instruments Ltd., Haverhill, UK).