Santacruzamate a

MSU crystals were produced in-house from urate (Sigma) and sodium hydroxide (Merck) as described previously [11 (link)]. Palmitic acid (Sigma) dissolved in 100% ethanol and human albumin (Albuman 200 g/L, Sanquin, Amsterdam) were conjugated as described previously [11 (link)]. Romidepsin, entinostat, santacruzamate A, RGFP966, and bortezomib were purchased from Selleckchem. Etomoxir was purchased from Sigma. HDAC6 inhibitor ITF3107 was kindly provided by Italfarmaco SpA, Milan, Italy.

Xenopus laevis were cared for and used according to approved IACUC and AAALAC protocols. Xenopus egg extracts and sperm chromatin were prepared as described previously (82 (link)). In brief, NPE was formed by incubating demembranated sperm chromatin in crude egg extract to form nuclei. Nuclei were isolated by centrifugation and then fractionated by ultracentrifugation to remove lipids and chromatin. The pActin, pActin^ΔTATA, pCMV, and pBRCA1 plasmids were constructed as described previously (35 (link)). For all reactions, extracts were supplemented with 1 mM DTT and ATP regenerating mix (6.5 mM phosphocreatine, 0.65 mM ATP, and 1.6 μg/ml creatine phosphokinase). Prior to the addition of DNA (T = 0 min), extracts were incubated at 21 °C (room temperature [RT]) for 10 min. Unless otherwise indicated, reactions were supplemented with 10 ng/μl plasmid DNA or 1250 demembranated sperm chromatin/μl. Where indicated, reactions were also supplemented with 10 to 125 μM HDAC inhibitors: SAHA (Cayman 10009929), Romidepsin (Selleckchecm S3020), RGFP966 (Selleckchem S7229), Santacruzamate A (Selleckchem S7595), 300 μM JQ1 (Sigma SML1524), 20 ng/μl ⍺-amanitin, 2 to 20 μM rNTPs, 0.5 ng/μl RNase A (Fisher), and/or 4 units/μl RNasin (Promega). All experiments were performed with at least two biological replicates and representative or average data are shown.