Dnmt activity inhibition assay

At an age of thirteen weeks, brains from 8 male and 9 female mice prenatally exposed to Poly I:C, and 9 male and 9 female offspring from PBS-treated dams were used for epigenetic analysis. For this purpose, mice were anesthetized with a single intraperitoneal injection of Ketamin (Pfizer, Germany) and Xylacin (Bayer, Germany). 0.7 ml 10 % Ketamin and 0.3 ml 2 % Xylacin were mixed and administered at a dose of 10 ml/kg. Afterwards, brains were collected and stored at −20 °C in RNA-Later (Invitrogen, Germany). Half of the brain was used to extract nuclear proteins using a Nuclear Extract Kit (Active Motif, Belgium) and the activities of various HDAC and DNMT enzymes were analyzed with the HDAC Fluorescent Assay Kit and DNMT Activity/Inhibition Assay (Active Motif, Belgium). The fluorescence/optical density was detected with a PolarStar OPTIMA microplate fluorimeter (BMG Labtech).

To assess activity of DNMT enzymes during in vitro spermatogenesis with exposure to different doses of PBB153, nuclear extracts were isolated using Nuclear Extraction kit per manufacturer’s instructions (Active Motif). To examine the activity of the DNMT enzymes, the isolated nuclear extracts were used in the DNMT Activity/Inhibition Assay per manufacturer’s instructions (Active Motif). The activity of the de novo DNMT enzymes was obtained by incubating the nuclear extracts in Complete Enzymatic Buffer AM1 (Active Motif) for 2 hours while the activity of the maintenance DNMT enzymes was obtained by incubating for 1 hour in accordance with manufacturer’s instructions. To directly examine the effect of PBB153 on the DNMT enzymes, PBB153, at a final concentration of 10 μM, was added to control nuclear extract prior to the initial incubation step. DNMT activity was determined by following the equation provided by the manufacturer (Active Motif). N = 4 in Fig. 3A,B, where each n represents an average of 6 measurements from the nuclear extract obtained from individual differentiations and n = 6 sample points in Fig. 3C,D where each n represents six replicates obtained from one differentiation.

DNMT enzyme activity of cell lines and xenograft tumors was evaluated using the DNMT Activity/Inhibition Assay (Active Motif) according to the manufacturer's instructions. Nuclear extracts were prepared by the Nuclear Extract Kit (Active Motif). Each 10 μg of nuclear extract was incubated with the enzymatic buffer containing AdoMet (1:100 dilution) for 2 h, and incubated with His‐MBD2b protein (1:50 dilution) for 45 min. followed by incubation for 45 min with anti‐polyHis‐HRP antibody (1:1000 dilution). The developing solution was added to each extract and the optical density was measured on a microplate reader at 450 nm.²⁵