Comet assay kit

Comet assay was performed following the instructions of the comet assay kit (KeyGEN BioTECH, Jiangsu, China) to make 3 layers of gel on the slides. The first layer was 0.5% normal melting point agarose; the second layer was 0.7% low melting agarose, and the third layer was 0.7% low melting agarose. The slides were lysed in lysis buffer for 2 h, and then immersed in an alkaline electrophoresis solution to untwist for 30 min. After single-cell electrophoresis, the slides were stained with propidium iodide (PI) for 10 min, and images were obtained with a laser confocal microscope (Leica, Solms, Germany). Tail DNA, tail length and olive tail moment (OTM) were analyzed through Comet Assay Software Project (CASP) [23 (link)].

DNA damage was detected with a comet assay kit (KeyGEN, Nanjing, China) under alkaline conditions. In brief, cells were harvested and washed with cold PBS buffer at indicated time points after 8 Gy X-ray irradiation. Cell concentration was adjusted to 1 × 10⁶ cells /mL using PBS. Then, mixed 10 μL glioma cells with 75 μL 0.7% low melting point agarose and plated the mixture on pre-coated normal melting point agarose slides. Slides were placed in pre-cooled lysis buffer at 4 °C for 2 h and transferred to the alkaline electrophoresis solution (1 mM EDTA, 300 mM NaOH) in a horizontal electrophoresis tank for 40 min at room temperature. Subsequently, the slides were electrophoresed at 25 V for 30 min and neutralized three times with 0.4 mM Tris-HCl (pH7.5) at 4 °C for 10 min each. Cells were finally stained with 20 μL Propidium Iodide for 10 min in the dark and observed under a Leica inverted fluorescence microscope. DNA damage was quantified by the comet tail intensity (tail DNA%) using Comet Assay Software Project (CASP, version 1.2.2).

Comet assay was performed using the Comet Assay kit (KEYGEN BIOTECH. CO., Nanjing, China) according to the manufacture’s instruction. After treatment with 1, 5, 10 μM of lapachol for 48 h, the cells were harvested and resuspended in 1 mL ice-cold PBS. Then 10 μL cell suspension (10⁴ cells) were mixed with 75 μL low-melting agarose at 37 °C for 20 min, and then added to clean microscope slides, which had been covered with 100 μL 0.75% normal-melting agarose, and the gels were solidified at 4 °C for 10 min. Then the slides were lysised for 1-2 h, immerged in alkaline buffer (1 mM EDTA and 300 mM NaOH), and then subjected to electrophoresis at 25 V for 30 min. Finally, 2 μL PI was dropped onto each slide and covered with a clean cover slip and then observed by a fluorescent microscope. A total of 50 C6 cells were randomly analyzed with an image analysis system (Komet 5.5, Kinetic Imaging Ltd., UK) and DNA migration was determined by measuring the “tail intensity” (% tail DNA).

Alkaline comet assay was performed using the comet assay kit according to the manufacturer’s protocol (KeyGEN Biotech, China). After the MDA-MB-231 cells were treated with different concentrations of BKM120, olaparib or their combination for 72 h, the cells were digested from the dishes and washed twice by Ca²⁺-free PBS. Untreated cells served as control. The cells (1 × 10⁴/ml) mixed with low melting point agarose at a ratio of 1:10 (v/v) were layered onto the Slide. The in gel cells were lysed by the lysis buffer at 4 °C for 2 h and then were unwound by alkaline unwinding solution for another 30 min at room temperature. Following an electrophoresis at 21 V for 30 min, the cells were stained with PI and were observed with the inverted biological microscope (Olympus BX53, Tokyo, Japan). Five images were randomly captured per slide. Nuclei were analyzed randomly 50 each from 3 slides per treatment and expressed as percent of tail DNA. The percentage of tail DNA [tail DNA (%)] was selected among the comet parameters as it gives us a clear indication of the extent of DNA damage induced by the test chemical^{39 (link)}.

The apoptosis detection kit, cell cycle detection kit and comet assay kit were bought from KeyGen Biotech company (Nanjing, China). MTT (Thiazolyl blue tetrazolium bromide) and DAPI (DAPI, dihydrochloride) were obtained from Sigma Chemical Co. (MO, USA). Antibodies against GADD45A, ERK, p-ERK and β-actin were obtained from Proteintech Group Inc. (Chicago, USA). Antibodies against γH2AX was from Cell Signaling Technology (MA, USA) while the enhanced chemiluminescence (ECL) kit was purchased from Amersham Life Science, Inc. (United States). A specific inhibitor of MEK1/2 (PD0325901) was purchased from Selleck (Shanghai, China).

Lapachol [2-hydroxy-3-(3 -methyl-2 -butenyl-)-1,4- naphthoquinone] was purchased from Sigma Aldrich (St. Louis, MO, USA). DMEM medium and fetal bovine serum were purchased from Invitrogen Co (Carlsbad, CA). 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H -tetrazolium (MTS)/phenazinemethosulfate (PMS) assay kit and Annexin V-FITC/PI staining kit were purchased from Promega (Madison, WI, USA). Comet assay kit was bought from Nanjing KeyGEN BioTECH. Co. Ltd. in China. Topoisomerase I and II drug screening kits were bought from Topogen Inc. (USA). Enzyme-linked Immunosorbent Assay Kits for Topoisomerase I (TOP I) and TOP II were bought from Cloud-clone Corp. (Houston, USA). All other chemicals used were of the highest purity available from commercial sources.