Trichostatin a tsa

For each sample, 1x10⁶ cells were transfected with the appropriate construct and 48hr later, subjected to immunoprecipitation using the relevant antibody. For experiments measuring the effect of Ins(1,4,5,6)P4, Ins(1,4,5,6)P4 (Cayman Chemical) was added to a final concentration of 12.5μM δυρινγ ιμμuνοπρεχιπιτατιον; immunoprecipitates were collected on either Protein A or Protein G beads. HDAC assays consisted of immunoprecipitates, HDAC buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 1mM MgCl₂), 5,000 cpm labeled histones, with or without 5μM trichostatin A (TSA; Sigma-Aldrich), in a final volume of 200μl. Samples were incubated at 37°C for 2hr on a half rotation, after which the reaction was terminated by the addition of 50μl Stop buffer (1M HCl/160mM acetic acid for labeled histones). Released ³H-acetate was extracted into 600μl ethyl acetate (Sigma-Aldrich) and the radioactivity determined using a liquid scintillation spectrometer (Beckman LS6500). Statistical analysis was performed using one-way ANOVA with post-hoc Tukey HSD.

Twenty gastric cancer cell lines, ten colorectal cancer cell lines, and two non-gastrointestinal cancer cell lines were maintained in high-glucose DMEM with 10% fetal calf serum (Gibco/Invitrogen, Carlsbad, CA) at 37°C in a humidified 5% CO₂ atmosphere. Names of used cell lines and histological types of gastric cancer cell lines were described in our previous reports [6] (link). Human T98G and A172 cell lines were purchased from the RIKEN Bio Resource Center (Tsukuba, Japan). For the treatment with DNA demethylation reagent or histone deacetylase (HDAC) inhibitor, 5-Aza-2′-deoxycytidine (5-Aza-dC, Sigma-Aldrich) at 2 µg/ml and/or trichostatin A (TSA, Sigma-Aldrich) at 25–1000 ng/ml were added to the culture medium.

To evaluate the role of HDAC1 and HDAC2 in uraemic endothelial dysfunction, a pan‐HDAC inhibitor trichostatin A (TSA) (Sigma Aldrich) and DF were used as HDAC inhibitors. ECs on 6‐well microplates were pretreated (24 hours) with TSA (50 nmol/L) or DF (100 µg/mL) and exposed to media containing 20% of pooled sera from the uraemic patients or healthy donors, as previously described for the differential proteomic analysis. Cells were then fixed and labelled for intercellular adhesion molecule‐1 (ICAM‐1) (Millipore, Merck), Toll‐like receptor 4 (TLR4) (Abcam) and von Willebrand Factor (vWF) (DAKO) using IF, as previously described. Reactive oxygen species (ROS) production was explored by using CM‐H2DCFDA. This compound can passively diffuse into cells being oxidized by ROS to the highly fluorescent dichlorofluorescein. ROS production was monitored by fluorescence microscopy. All samples were evaluated by microscopy (Leica DM4000B), and images were captured through a videocamera (Leica DFC310FX). Fluorescence micrographs were analysed using ImageJ (version 1.43m; NIH, http://rsb.info.nih.gov/nih-image/manual/tech.html#). Cultured cells were selected from the background with the threshold tool, and the fluorescence intensity was measured.