Acridine orange base

NL-101 was designed and synthesized by Hangzhou Minsheng Institute of Pharmaceutical Research. Dimethyl sulfoxide and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (USA). Propidium iodide (PI)/RNase staining kit and Annexin V-FITC/7AAD kit were purchased from Becton Dickinson (San Diego, CA, USA). Acridine Orange base was purchased from Sigma-Aldrich (USA). Agarose was purchased from Invitrogen (USA). Low-melting Agarose was purchased from Sangon Biotech (Shang Hai, China). Antibodies against p21 (ab18209), γ-H2AX (ab11134), were purchased from Abcam (Cambridge, UK). Antibody against acetylated histone H3 was from Merck Millipore. Antibodies against histone H3 (db972), H2AX (db5729), and Chk2 (db928) were from Diagbio, and antibodies against caspase-3 (#9662), cleaved caspase-3 (Asp175, #9664), caspase-8 (#4790), caspase-9 (#9508), cleaved caspase-9 (Asp330, #9501), PARP (#9532), c-Myc (#9402), cyclin E1 (#4129), Cdk2 (#2546), ATG5 (#2630), SQSTM1/p62 (#5114), LC3B (#3868s), p-mTOR (#5536), mTOR (#2938), p-Akt (#4060), Akt (#4691), p-Chk2 (#12302), and β-actin (#4970) were purchased from Cell Signaling Technologies (Danvers, MA, USA). And horseradish peroxidase-conjugated secondary antibodies were purchased from Bio-Rad (Hercules, CA, USA).

A stock acridine orange base (Sigma-Aldrich, 235474) solution of 2.5 mg/mL in dimethyl sulfoxide (DMSO) was made and stored at -20 °C until used. At 24 h, 36 h and 48 h acridine orange stock solution was added to embryo medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂ · 2H₂O and 0.33 mM MgSO₄ · 7H₂O in water) to make a final concentration of 5 μg/mL. Embryos were bathed in the acridine orange / embryo medium solution in the dark at 28.5 °C for 28 min. Embryos were then washed five times in embryo medium for 5 min each and analyzed using fluorescent microscopy on a Zeiss Axio Imager M1 compound microscope and Olympus SZX16 dissecting microscope.