(Z)-Mirin is a potent Mre11–Rad50–Nbs1 (MRN) complex inhibitor, and inhibits Mre11-associated exonuclease activity.

Mirin抑制由DSB引起的ATM激活、ATM依赖的下游靶点Nbs1和Chk2的磷酸化以及对DSB反应中的MRN依赖的ATM在Ser1981的自磷酸化。Mirin还抑制TOSA4细胞中的G2检查点和HEK293细胞中的同源依赖DNA修复。[1] 在集成了HPV16的细胞（SiHa）中，Mirin使HPV episomes对PA25敏感，导致PA25 IC50减少约5倍。[2] Mirin的预处理还降低了顺铂处理的人胚肾293细胞的存活率，抑制了增殖细胞核抗原表达。[3]

Nuclease assay: Reactions with oligonucleotide nonhairpin substrates contains 25 mM MOPS (pH 7.0), 60 mM KCl, 0.2% Tween 20, 2 mM DTT, 1 mM or 5 MnCl2 (or 5 mM MgCl2, or 5 mM CaCl2), 0.1 pmol of DNA substrate, and 0.3 pmol of Mre11 (or an equivalent amount of Mre11 complexed with Rad50) in a volume of 10 μl, and are incubated at 37°C for 30 min. SDS, EDTA, and proteinase K are then added to final concentrations of 0.2%, 5 mM, and 0.1 mg/ml, respectively, and incubated for another 15 min. 4 μl of each reaction is mixed with 4 μl of formamide loading buffer, and then loaded onto a sequencing gel containing 10% acrylamide and 7 M urea. After the run, each gel is analyzed using a phosphorimaging system. Reactions containing hairpin substrates are identical to those with nonhairpin substrates except that 3 pmol of Mre11 is added to reactions as indicated, and the reactions are incubated at room temperature overnight. Nonhomologous end-joining reactions contains 25 mM MOPS (pH 7.0), 60 mM KCl, 0.2% Tween 20, 2 mM DTT, 4 mM MgCl2, 2 mM MnCl2, 0.5 mM ATP, 4 ng of plasmid DNA, 10% polyethylene glycol, 0.01 pmol of human DNA ligase I, and 0.06 pmol of Mre11 or 0.1 units of E. coli exonuclease III (GIBCO-BRL), in a volume of 10 μl. After incubation at 37°C for 25 min, Tween 20 is added to a final concentration of 0.5%, and a 2.5 μl aliquot is amplified by PCR using primers DAR5 and DAR147. PCR products are cloned using the TA cloning kit and sequenced using an automated ABI Capillary Genetic Analyzer.

Human embryonic kidney (HEK) 293 cells are maintained in RPMI-1640 supplemented with 5% heat-inactivated fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 mg/mL) in humidified air with 5% CO2 at 37 °C. Cells are given fresh medium at 48 h intervals. The cells are seeded in 96-well plates in regular growth medium. Cells are pretreated with mirin (100 μM for 1 h before the cisplatin (20 μM) treatment followed by incubation for 8 and 24 h. The MTT assay is performed using the EZ-Cytox cell viability assay kit according to the manufacturer's protocol and MTT reduction is measured at a 450 nm wavelength using a micro-plate reader. (Only for Reference)