Panobinostat inhibits the levels and activity of HDAC7 as well as induces Nur77 and Nor1 levels. (A) HH and MJ cells were treated with the indicated concentrations of panobinostat for 4 hours. Following this, total RNA was isolated and RT-PCR was done for HDAC7, Nur77, and Nor1. A β-actin–specific PCR and expression served as the loading control for the amplification. Alternatively, after treatment with panobinostat, total cell lysates were isolated and immunoblot analysis was done for HDAC7, Nur77, and acetylated α-tubulin. The levels of β-actin in the cell lysates served as the loading control. (B) Recombinant HDAC7 (3.6 μg protein/well) was incubated at 37°C with 100 μM Fluor de Lys substrate (BIOMOL Research Laboratories, Plymouth Meeting, PA) and indicated concentrations of panobinostat (0, 5, 10, 20, and 50 nM) for 1 hour in the assay buffer provided by the manufacturer. Reactions were stopped with Fluor de Lys developer and fluorescence was measured (CytoFluor II; PerSeptive Biosystems [BioTek Instruments, Winooski, VT]; excitation: 360 nm, emission: 460 nm). Experiment was performed in triplicate and the value of the blank was deducted from all experimental values. (C) FLAG-tagged HDAC7 was immunoprecipitated from untreated HH cells. Fifteen micrograms (1 μg/μL) HDAC7 immunoprecipitate was incubated at 37°C with 100 μM Fluor de Lys substrate and indicated concentrations of panobinostat for 1 hour in the assay buffer provided by the manufacturer. Reactions were stopped with Fluor de Lys developer and fluorescence was measured (CytoFluor II; excitation: 360 nm, emission: 460 nm). The value of the blank was deducted from all experimental values. Values represent the mean ± SEM of 3 experiments.