Trichostatin a

Male C57Bl6/N-Plp^null/y (Plp^null/y) mice and their corresponding wild-type male littermates were sacrificed at the age of 75 days. A light weighted membrane fraction enriched in myelin was obtained from frozen half brains by sucrose density gradient centrifugation essentially as described previously [51 (link)]. Briefly, after homogenizing the brains in 0.32 M sucrose solution containing protease (cOmplete, Roche) and deacetylase inhibitors (10 mM nicotinamide, 0.5 μM Trichostatin A, Sigma), a first fraction enriched in myelin was obtained by density gradient centrifugation on a 0.85 M sucrose cushion. After washing and 2 consecutive osmotic shocks, the final brain myelin fraction was purified by sucrose gradient centrifugation as before. The myelin fraction was washed and suspended in TBS buffer (137 mM NaCl, 20 mM Tris/HCl [pH 7.4], 4°C) supplemented with protease (cOmplete, Roche) and deacetylase inhibitors (10 mM nicotinamide, 0.5 μM Trichostatin A, Sigma). Protein concentration was determined using the DC Protein Assay (Bio-Rad) according to the manufacturer’s instructions.

HAA1 cells were plated onto 60 mm plates at a density of 8.0 × 10⁵ in RPMI 1640 medium supplemented with 10% FBS. Then, 24 h after plating, cells were either left untreated or treated with sodium butyrate (3 mM, Thermo) or trichostatin A (100–300 nM, Sigma-Aldrich, Saint Louis, MO, USA). Cells were maintained in the presence of the HDACi for a defined number of days, at which time the media was removed and cells harvested directly on the plate with TRI^® reagent (Sigma-Aldrich, St. Louis, MO, USA). Experiments with HDAC isoform-specific inhibitors and suberoylanilide hydroxamic acid (SAHA) were performed in a similar fashion, except that HAA1 cells were grown in triplicate in 6-well plates (Corning) and either left untreated or treated for 4–6 days with sodium butyrate, trichostatin A, SAHA (5 µM, #SML0061, Sigma-Aldrich, St. Louis, MO), CI994 (50 µM, #S2818, SelleckChem, Houston, TX, USA), PC-34051 (50 µM, #S2012, SelleckChem) or RGFP996 (50 µM, #S7229, SelleckChem, Houston, TX, USA). RNA from untreated and treated cells was prepared and analyzed as described below.

ESCC cell lines were treated with 10 μmol/L 5-aza-2V-deoxycytidine (a demethylation agent) (Sigma-Aldrich, Steinheim, Germany) as previously described 28 (link). For the treatment combining 5-aza-2V-deoxycytidine and trichostatin A (Sigma-Aldrich), cells were treated with 5-aza-2V-deoxycytidine for 3 days and subsequently treated with trichostatin A (an inhibitor of histone deacetylases) (100 ng/mL) for 24 hours.

U2-OS, MCF7, IMR90, HCT116, A549, and MEF cells were maintained in DMEM supplemented with 10% (12% for MEFs) FBS and 1% penicillin/streptomycin sulfate (Invitrogen Inc., Carlsbad, CA, USA) at 37 °C in a humidified incubator under 5% CO₂. Prior to IGF-1 treatment, MCF7 and HCT116 cells at 60–70% confluency were incubated in serum-free DMEM for 48 h, while MEFs and IMR90 cells were serum-starved in DMEM containing 0.5% FBS. IGF-1 (human recombinant; Sigma, St. Louis, MO, USA) was prepared as a 100 μg mL⁻¹ stock solution in PBS according to manufacturer’s instructions. LY294002 (Calbiochem, San Diego, CA, USA) was prepared as a 25 mm stock solution. Trichostatin A (TSA; Sigma) was prepared as a 2 mg mL⁻¹ stock in DMSO. At 45 min prior to IGF-1 treatment, LY294002 was added to serum-starved cells at a final concentration of 25 μm. For acetylation of endogenous p53, cells were deprived of serum for 48 h before treatment with IGF-1 for 12 h and subsequently treated with 40 μm Trichostatin A for 6 h. Nutlin-3a (Sigma) was used at 5.0 nm either with IGF-1 or with the vehicle control.

A549 cells (10 cm dishes) were treated with vehicle or SBI-797812 (10 μM) for 4 h as described above. Where indicated, 0.5 mM H₂O₂ was also added to the cells for 30 min (from 3.5 to 4 h). Cell lysis buffer contained RIPA, NAM (10 mM), trichostatin-A (10 μg ml⁻¹; Sigma-Aldrich), protease inhibitor cocktail (1×, Roche), olaparib (5 μM; Cayman Chemical), ADP-HPD (250 nM; Millipore Sigma) and benzonase nuclease (0.2 U μl⁻¹; Sigma-Aldrich). Where indicated (Fig. 5c), olaparib and ADP-HPD were excluded during the cell lysis step. Samples were probed by western blotting with the following antibodies: rabbit monoclonal anti-PARP1 (Cell Signaling, cat no. 9532, clone 46D11) 1:1000 dilution, rabbit polyclonal (affinity-purified) anti-PAR (Trevigen 4336-APC-050) 1:1000-dilution, rabbit monoclonal anti-histone H4 (Cell Signaling, cat no. 13919, clone D2X4V) 1:1000-dilution, and rabbit polyclonal (affinity-purified) anti-acetyl-histone H4(Lys16) (Millipore Sigma, cat no. 07-329) 1:5000-dilution. Protein bands were visualized with IRDye-labeled secondary antibodies (LI-COR Biosciences) followed by scanning with the Odyssey Digital Infrared Imaging System (LI-COR Biosciences).