Dnmt activity inhibitor assay kit

All compounds were screened for total DNMT activity using a DNA methyltransferase (DNMT) activity/inhibitor assay kit (Epigentek, Farmingdale, NY, USA) according to manufacturer's instruction. To measure the effects of the compounds on DNMT activity, 35 μg of total HaCaT cellular extract freshly prepared in RIPA buffer (Tris-HCl pH 7.4 10 mm, NaCl 150 mm, NP-40 1%, sodium deoxycholate (DOC) 1%, SDS 0.1%, glycerol 0.1%, Protease Inhibitors Cocktail) was incubated with 50 and 100 μm of the different compounds in DMSO (1% final conc.) or vehicle alone (1% DMSO) at 37 °C for 2 h. As negative control, the lysate was denatured at 100 °C for 30 min. Total lysate was used to avoid the loss of possible coenzyme and complex formation. The amount of methylated DNA, which is proportional to enzyme activity, was colorimetrically detected by a plate reader at 450 nm (EnSpire ® Multimode Plate Reader -Perkin Elmer).

Compounds 1, 22 and 24 were tested to evaluate their selectivity on the different DNMT isoforms. To work selectively on DNMT1, DNMT3A, and DNMT3B, HEK293T cells were transfected with the plasmids containing the three different DNMTs' sequences and mock control. Cells were transfected with 2.5 μg of expression plasmid using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) according the manufacturer's instruction. Plasmids pcDNA3/Myc-DNMT1 (Addgene plasmid # 36939), pcDNA3/Myc-DNMT3A (Addgene plasmid # 35521), and pcDNA3/Myc-DNMT3B1 (Addgene plasmid # 35522) were a gift from Arthur Riggs. [39, 40] The presence of exogenous DNMTs was checked by Western blot (not shown), and afterward, the transfected cells were freshly lysed in RIPA buffer as above. Cellular extract of 35 μg were incubated with selected compounds at different concentration in the 1-150 μm range (1% DMSO final conc.) or with vehicle alone (1% DMSO) at 37 °C for 2 h. RG108 (Cayman) was used as positive controls, while as negative control, lysates were denatured at 100 °C for 30 min. DNMT activity was detected by DNMT activity/inhibitor assay kit (Epigentek). Data are presented as means ± SD; each compound was tested at least three times.