Enzyme inhibition assays are performed using the Reaction Biology HDAC Spectrum platform. The HDAC1, 2, 4, 5, 6, 7, 8, 9, 10, and 11 assays use isolated recombinant human protein; HDAC3/NcoR2 complex is used for the HDAC3 assay. Substrate for HDAC1, 2, 3, 6, 10, and 11 assays is a fluorogenic peptide from p53 residues 379-382 (RHKKAc); substrate for HDAC8 is fluorogenic diacyl peptide based on residues 379-382 of p53 (RHKAcKAc). Acetyl-Lys (trifluoroacetyl)-AMC substrate is used for HDAC4, 5, 7, and 9 assays. Tubastatin A is dissolved in DMSO and tested in 10-dose IC₅₀ mode with 3-fold serial dilution starting at 30 μM. Control Compound Trichostatin A (TSA) is tested in a 10-dose IC₅₀ with 3-fold serial dilution starting at 5 μM. IC₅₀ values are extracted by curve-fitting the dose/response slopes.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Primary cortical neuron cultures are obtained from the cerebral cortex of fetal Sprague-Dawley rats (embryonic day 17). All experiments are initiated 24 hours after plating. Under these conditions, the cells are not susceptible to glutamate-mediated excitotoxicity. For cytotoxicity studies, cells are rinsed with warm PBS and then placed in minimum essential medium containing 5.5 g/L glucose, 10% fetal calf serum, 2 mM L-glutamine, and 100 μM cystine. Oxidative stress is induced by the addition of the glutamate analogue homocysteate (HCA; 5 mM) to the media. HCA is diluted from 100-fold concentrated solutions that are adjusted to pH 7.5. In combination with HCA, neurons are treated with Tubastatin A at the indicated concentrations. Viability is assessed after 24 hours by MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

The effects of HDAC6 targeting in dextran sodium sulfate (DSS) and adoptive transfer models of colitis are evaluated, using 10 mice per group. Freshly prepared 4% (wt/vol) DSS is added daily for 5 days to the pH-balanced tap water of WT B6 mice. Mice are treated daily for 7 days with tubacin or niltubacin (0.5 mg/kg of body weight/day, i.p.), and colitis is assessed by daily monitoring of body weight, stool consistency, and fecal blood. Stool consistency is scored as 0 (hard), 2 (soft), or 4 (diarrhea), and fecal blood (Hemoccult) is scored as 0 (absent), 2 (occult), or 4 (gross). To assess prevention of colitis in a T cell-dependent model, CD⁴⁺ CD45RBhi T cells (1×10⁶) isolated from WT mice using magnetic beads (>95% cell purity, flow cytometry) are injected i.p. into B6/Rag1^?/? mice plus CD⁴⁺ CD²⁵⁺ Tregs (1.25×10⁵) isolated using magnetic beads from HDAC6^?/? or WT mice (>90% Treg purity, flow cytometry) and mice are monitored biweekly for clinical evidence of colitis. To assess therapy of established T cell-dependent colitis, B6/Rag1^?/? mice are injected i.p. with CD⁴⁺ CD45RBhi cells (1×10⁶). Once colitis has developed, mice also receive CD⁴⁺ CD²⁵⁺ Tregs (5×10⁵ cells) isolated as described above from HDAC6^?/? or WT mice or treatment with HDAC6i (tubastatin A) or HSP90i (17-AAG). Mice are monitored for continued weight loss and stool consistency. At the cessation of the study, paraffin sections of colons stained with Alcian Blue or hematoxylin and eosin are graded histologically or evaluated by immunoperoxidase staining for Foxp³⁺ Treg infiltration.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

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