* Correspondence to: Richard Bayles; Email: richard. bayles@ bakeridi. edu. au Submitted: 10/06/10; Accepted: 10/06/10 Previously published online: www. landesbioscience. com/journals/cc/article/13888 DOI: 10.4161/cc. 9.22. 13888 demonstrated an increase in NET gene transcription in response to depolarisation with potassium. 3 Contrary to BDNF gene regulation, which associated MeCP2 binding with DNA methylation levels of the promoter, in that report we demonstrated for the first time that the transcriptional increase of the NET gene occurred in the absence of changes in promoter methylation but, rather, was associated with changes in histone modifications. A number of conditions, including depression, 4 are characterized by a reduction in NET activity and pharmacological agents, such as valproic acid, have the capacity to inhibit Class I HDACs. 5 The potential for HDAC inhibitors to increase NET gene expression merits investigation. Cortical neurons from the cerebral neocortex were cultured from C57Bl6 embryonic mice (E15) as described previously. 3 Briefly, cells were seeded at 200,000 cells/cm2 and maintained in a humidified 37 C incubator under 5% CO2. Twenty-four hours after seeding, cultures were maintained in serum-free Neurobasal medium containing B27 supplement (Invitrogen, CA USA). At DIV4 cells were then treated with a range of concentrations (0.3 mM, 0.6 mM and 1.2 mM) of valproic acid sodium salt (Sigma-Aldrich, MO USA) or the structurally related (but lacking HDAC inhibitory activity) valpromide (Alfa Aesar, MA USA) for 16 hours, or the more potent Class I and II HDAC inhibitor Trichostatin A (TSA)(Sigma-Aldrich) at 100 ng/mL for 8 hrs, using DMSO or media controls where appropriate. Cell viability was assessed by the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, WI USA). No significant difference in cell viability was observed for any treatment used (data not shown).

Total RNA was prepared using the Trizol method (Invitrogen) and cDNA was prepared using the SuperScript® III First-Strand Synthesis System (Invitrogen). Quantitative RT-PCR was performed using SYBR® Green JumpStart™ Taq ReadyMix™(Sigma-Aldrich). Expression of the NET gene was calculated relative to β-actin gene expression, which remained stable under all conditions tested. Chromatin immunoprecipitation (ChIP) assays were performed as described previously, 6 with some modification using Dynabeads® Protein G (Invitrogen) and the following antibodies (Abcam, MA USA) of interest: H3K9m2 (ab1220), H3K9m3 (ab8898), H3K9/K14ac (ab06-599). After washing of immune complexes, DNA was recovered using the MinElute Reaction Clean-up Kit (Qiagen, Hilden, Germany). Analysis of ChIP DNA samples was performed by quantitative PCR (qPCR) of the NET gene promoter region. An input sample was used as an internal control for variation in total DNA between samples. Further details and all primer sequences are available on request.