Hdac3

A549, H1299 and H460 human lung cancer cells purchased from the American Type Culture Collection (Manassas, VA, USA), Lu99 human lung cancer cells, purchased from the RIKEN cell bank (Tsukuba, Japan), and HCT 116 colorectal cancer cells (p53 null and p53 wild) were supplied by Dr. Kee-Ho Lee (KIRAMS, KOREA) were grown in the recommended growth medium (Invitrogen, Carlsbad, CA, USA). SAHA was purchased from ALEXIS Corporation (Lausen, Switzerland). Antibodies against HDAC1, HDAC2, HDAC3, cIAP2, Mdm2, HA, Myc and β-actin were acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HDAC4, SIRT1, SIRT2, histone 3, acetyl-histone 3, acetyl-histone 4, acetyl-p53 (Lys382), puma, ubiquitin, caspase 3, cleaved PARP, p-ATM, ATM, p-ATR, ATR, p-Chk1, Chk1, p-Chk2, Chk2, p-γH2AX, γH2AX and survivin antibodies were acquired from Cell Signaling Technology (Beverly, MA, USA). XIAP, caspase 7 and p21 antibodies were purchased from BD Biosciences Pharmingen (San Diego, CA, USA), and the p53 antibody was from Novocastra Lab. Ltd. (Newcastle, UK). The Flag antibody, Nutlin-3A and MG132 were from Sigma. (St Louis, MO, USA). The siRNAs targeting HDAC1, HDAC2, HDAC3, or HDAC4 were from Santa Cruz Biotechnology. Two different HDAC2 siRNAs (siHDAC #2 and siHDAC #3) and p53-specific siRNA were purchased from Ambion (Austin, TX, USA).

To measure the protein concentrations in the hippocampus, we homogenized the tissue in RIPA lysis buffer supplemented with a cocktail of protease and phosphatase inhibitors (Applygen, China) on ice. After centrifugation, the supernatant protein concentration was determined with a bicinchoninic acid (BCA) protein assay reagent kit (Thermo Pierce, USA). After determination of the protein concentration, protein samples were separated by SDS-PAGE and subsequently transferred to PVDF membranes (Bio-Rad, USA). Membranes were blocked with 5% nonfat milk in TBST (0.1% Tween-20 in TBS) and then incubated at 4 °C overnight with primary antibodies: HDAC2 (1:2000, Cell Signaling Technologies, USA), HDAC3 (1:2000, Santa Cruz, USA), HDAC4 (1:2000, Cell Signaling Technologies, USA), HDAC6 (1:4000, Merck Millipore, USA), Acetyl-H3 (1:2000, Merck Millipore, USA), Acetyl-H3K9 (1:2000, Merck Millipore, USA), Acetyl-H3K14 (1:2000, Merck Millipore, USA), BDNF (1:1000, Abcam, UK), c-Fos (1:2000, Merck Millipore, USA), and β-actin (1:2000, Santa Cruz, USA). After being rinsed with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:1000, Beyotime Institute of Biotechnology, China). Protein bands were visualized with an ECL detection system (Beyotime Institute of Biotechnology, China) and quantified with ImageJ software (NIH).

Proteins were resolved by SDS-PAGE and electrophoretically blotted onto Immun-Blot PVDF membranes (Bio-Rad, Hercules, California), followed by blocking in 5% nonfat milk. Primary antibodies (Abs) used included rabbit polyclonal anti-AQP5 (#AQP005, Alomone Labs, Jerusalem, Israel), -p300 (#SC584, Santa Cruz Biotechnology, Dallas, Texas), -HDAC2 (#SC7899, Santa Cruz Biotechnology) and -HDAC3 (SC11417, Santa Cruz Biotechnology). Protein was normalized to eukaryotic initiation factor 2α (eIF-2α), β-actin or GAPDH using rabbit anti-eIF2α (#11386, Santa Cruz Biotechnology) polyclonal Ab and mouse anti-β-actin (LMAB-C4, Seven Hills, Cincinnati, OH) and anti-GAPDH (#AM4300, Applied Biosystems, Foster City, CA) monoclonal Abs, respectively. Blots were incubated with horseradish peroxidase-linked anti-IgG conjugates (Promega) for 45 min at room temperature (RT). Complexes were visualized by enhanced chemiluminescence (ECL) (Thermo Scientific, Waltham, MA) with a FluorChem 8900 Imaging System (Alpha Innotech/Cell Biosciences, Santa Clara, CA).