Epiquik hdac activity inhibition assay kit

DNA extraction from mammary tumors of control, BSp, GTPs and combination treatment groups was described before. The global DNA methylation status was explicitly indicated by the levels of 5-methylcytosine (5-mC) in total DNA and was determined by the MethylFlash Methylated DNA 5-mC Quantification Kit from EpiGentek. In addition, the MethylFlash Hydroxymethylated DNA 5-hmC Quantification Kit (EpiGentek, Farmingdale, NY, USA) was employed to quantify global hydroxymethylation status in total DNA samples. The nuclear protein was extracted to determine overall DNMT and HDAC enzymatic activities using the EpiQuik DNMT Activity/Inhibition Assay Ultra Kit (EpiGentek) and the EpiQuik HDAC Activity/Inhibition Assay Kit (EpiGentek), respectively. Moreover, histone acetylation activities were evaluated using EpiQuik Acetyl Histone3-Lysine9 and Histone3-Lysine27 (H3K9 and H3K27) Assay Kits as per the manufacturer’s guidelines.

Cultured cells were harvested at the indicated time points and nuclear proteins from breast cancer MDA-MB-231 and MDA-MB-157 cells were extracted by NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific). The activities of HDACs and DNMTs were evaluated by EpiQuik HDAC Activity/Inhibition Assay Kit and EpiQuik DNMT Activity/Inhibition Assay Kit (Epigentek, Brooklyn, NY) according to the manufacturer’s protocols, respectively, as done previously^{24 (link)}. The enzymatic activities of HDACs and DNMTs were colorimetrically demonstrated and detected by an Epoch Microplate Spectrophotometer at 450 nm.

HuCCT-1 and HuH28 cells were treated with different concentrations of SFN (0–80 μM) or GEM (0–10 μM) for 3 h. To measure HDAC activity, nuclear extracts were obtained from cultured cells or 20 mg of subcutaneous tumor samples using an EpiQuik™ Nuclear Extraction Kit (Epigentek, Farmingdale, NY, USA) according to the manufacturer’s protocol. HDAC activity was measured in 10 μg of nuclear extract using an EpiQuik™ HDAC activity/inhibition assay kit (Epigentek) according to the manufacturer’s instructions. HAT activity was also measured in 10 μg of nuclear extract from cultured cells using an EpiQuik™ HAT activity/inhibition assay kit (Epigentek) according to the manufacturer’s instructions.
To detect total histone H3 and H4 acetylation, histone extracts were obtained from cultured cells using an EpiQuik™ Total Histone Extraction Kit (Epigentek). Histone H3 and H4 acetylation was detected in 100 ng of histone extract using an EpiQuik™ Total Histone H3 Acetylation Detection Fast Kit and an EpiQuik™ Total Histone H4 Acetylation Detection Fast Kit (Epigentek) according to the manufacturer’s instructions, respectively.
Dimethyl sulfoxide (DMSO, Nacalai Tesque, Inc.) was used as a vehicle, and HDAC activity and total histone H3 acetylation in cells treated with SFN and/or GEM were measured relative to that in the vehicle treatment group.

Nuclear protein extracts of P. falciparum NF54 were obtained as described before^{65 (link)} following the modifications by Anne Hempel from the Manuel Llinás group⁶⁶ . Briefly, P. falciparum NF54 cultures were pelleted (800 g, 5 min, low brake) and red blood cell lysis was promoted in a 0.1% PBS/saponin solution; parasites were then pelleted (800 g, 10 min, low brake) and lysed in 20 mM HEPES, pH 7.8, 10 mM KCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF and 1% Triton X-100. Nuclei were harvested by centrifugation at 2,500 g for 5 min and nuclear proteins were extracted for 30 min using 20 mM HEPES, pH 7.8, 800 mM KCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF and 1 × protease inhibitor cocktail (Sigma, P1860). Debris was removed by centrifugation (13,000 g, 30 min) and nuclear protein extract was kept at −80 °C in 15% glycerol until further use. HDAC inhibition by serial dilutions of romidepsin (AOBIOUS, AOB1853) or n-butanol bacterial culture supernatants in 20% DMSO was assessed using the colorimetric EpiQuik HDAC Activity/Inhibition Assay Kit (EpiGenTek, P-4002) according to the manufacturer’s protocol and using 10 µg of these P. falciparum nuclear protein extracts as source of Plasmodium HDAC enzymes.