Trichostatin a

Prostratin, Trichostatin A (TSA), and cyclosporin A are from Santa Cruz. Hexamethylene bisacetamide (HMBA) is from Sigma. MG-132 is from Boston Biochem. Bay 11-7082 is from Calbiochem. DyNAmo™ ColorFlash Master Mix is from Thermo Scientific. M-MLV is from Takara. All other chemicals are from AMRESCO or Sigma.

The 1,4-benzodiazepine ring (5-phenyl-1,3-dihydro-2-oxo-benzo[e][1,4]-diazepine) was used as the cap of novel hydroxamic-based HDACi [13 (link)]. (S) and (R) N1-hydroxy-N8-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]-diazepin-3-yl)octanediamide [(S)-8] and [(R)-8] were obtained as reported previously [16 (link)] where they are labelled with the number 8. The chiral compounds (S)-8 and (R)-8 (Fig. 1) were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St Louis, MO, USA) and stored as 0.1 M stock solutions in the dark at room temperature and added directly to the culture media. The amount of DMSO used as the vehicle did not interfere with drug activities. The antioxidant N-Acetyl-Cysteine (NAC, Sigma-Aldrich), the pan-caspase inhibitor Z-VAD-fmk (R&DSystems, Minneapolis, MN, USA), the phosphatase inhibitors Calyculin A and Okadaic acid, and the pan-deacetylase inhibitor trichostatin A (TSA; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were also used. The WST-1 reagent (Roche Diagnostic GmbH, Mannheim, Germany) was employed to assess cell proliferation in culture. All other chemicals were reagent grade.

HDAC activity assays were performed in 96-well opaque microplates as previously described29 (link). 20 μg of HeLa nuclear extract (Promega) or 50 ng recombinant Flag-HDAC 2 (BPS Bioscience) were diluted in assay buffer 1 (25 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂, and 1 mg/mL BSA) and incubated with the indicated amounts of pharmacological compounds, purified actin (Cytoskeleton Inc.) or BSA (Sigma-Aldrich) for 15 m at room temperature. Boc-L-Lys (AC)-AMC substrate in assay buffer 2 (25 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂) diluted to 25 μM was then added for 1 h. The reaction was stopped with 1 mg/mL trypsin (Sigma-Aldrich) and 5 μM trichostatin A (Santa-Cruz) in assay buffer 2. Plates were read using an excitation wavelength of 360 nm and an emission wavelength 460 nm.