4.2. Western Blot and HDAC Inhibitory Assay

When Saos-2 cells were grown, approximately 70% confluent, they were treated with 1, 10, and 100 µM 4HR for 2, 8, or 24 h; control cells were treated with 0.1% dimethyl sulfoxide in culture medium. Cultured cells were harvested with 0.01% trypsin and 1 mM EDTA. Cellular lysis was performed with a protein lysis buffer (PRO-PREPTM, iNtRON Biotechnology INC, Sungnam, Korea). Collected lysates were used for the Western blotting of HDAC1, HDAC3, HDAC4, HDAC5, Ac-lys, H3, and Ac-H3. Antibodies for HDACs and Ac-lys were purchased from SantaCruz Biotechnology (Santa Cruz, CA, USA). The quantification of the proteins was performed as previously described [22,23].

HDAC enzyme activity after 4HR administration was assessed by a commercially available kit (CAT: ab156064, Abcam, Cambridge, UK). Saos-2 cells received 1, 10, and 100 μM of 4HR, and cellular lysates were collected after 2, 8, and 24 h. The subsequent procedure was in accordance with the manufacturer’s protocol. The activity of HDAC1, 2, 3, and 8 (Class I HDAC) was measured with this kit according to the product datasheet. HDAC assay buffer and substrate were added to the reaction wells. Inhibitor and developer were placed into wells and thoroughly mixed. The prepared samples were added to each well and incubated for 20 min at room temperature. Then, the stop solution was added and incubated for 10 min at room temperature. Fluorescence intensity was measured with a plate reader.