The enzyme activity of HDAC class I and II was measured using the HDAC-Glo I/II assay kit (Promega, Madison, USA) according to the manufacturer's instruction. Briefly, 1-2×104 cells were suspended and cultured in a white-walled 96-well plate for 4 h, and three-fold serial dilutions (0.001, 0.002, 0.005, 0.014, 0.041, 0.123, 0.370, 1.111, 3.333, 10 µM) of MPT0B291 or SAHA dissolved in assay buffer were added and incubated at 37 °C for 30 min. The HDAC-Glo reagent was then added to each well, and the luminescence signal was measured after incubation at room temperature for 30 min.
HDAC6 enzyme activity was measured using a fluorometric HDAC6 activity assay kit (BioVision, CA, USA), according to manufacturer's instruction. Briefly, 1-2×106 cells were lysed in 100 μL of lysis buffer and centrifuged at 16000 ×g for 10 min. Two-fold serial dilution (0.020, 0.039, 0.078, 0.156, 0.312, 0.625, 1.25, 2.5, 5, 10 µM) of MPT0B291, SAHA, tubacin, and lysates were mixed and incubated in white-walled 96-well plates at 37 °C for 10 min. The HDAC6 substrate was then added and the fluorescence signal (excitation/emission = 380/490) was measured after incubation at 37 °C for 30 min 15 (link).
HDAC6 enzyme activity was measured using a fluorometric HDAC6 activity assay kit (BioVision, CA, USA), according to manufacturer's instruction. Briefly, 1-2×106 cells were lysed in 100 μL of lysis buffer and centrifuged at 16000 ×g for 10 min. Two-fold serial dilution (0.020, 0.039, 0.078, 0.156, 0.312, 0.625, 1.25, 2.5, 5, 10 µM) of MPT0B291, SAHA, tubacin, and lysates were mixed and incubated in white-walled 96-well plates at 37 °C for 10 min. The HDAC6 substrate was then added and the fluorescence signal (excitation/emission = 380/490) was measured after incubation at 37 °C for 30 min 15 (link).