Attractene

Cell culture reagents were purchased from GE and fetal bovine serum (FBS) was purchased from PAN-Biotech; DTT was purchased from Millipore (Massachusetts, USA); Compound C, AICAR, Resveratrol, Suramin, SNAP, and ODQ were purchased from Sigma-Aldrich; N6022, Pifithrin-α, VO-Ohpic trihydrate, AS1842856, and MK 2206 were from MCE. Hydrogen peroxide (H₂O₂) was from Sinopharm Chemical Reagent; 3-(4,5-dimethyl-2-thiazolyl)−2,5-diphenyl-2-H-tetrazolium bromide (MTT) was purchased from Sangon Biotech; Lipofectamine® 3000 and Attractene were from Thermo Fisher Scientific and Qiagen, respectively. Fluor-de-Lys SIRT1 fluorometric drug discovery assay kit was from Enzo Life Sciences; Hydrogen Peroxide Assay Kit, Nitric Oxide Assay Kit, RIPA lysis buffer, Bicinchoninic Acid assay Kit, and Nuclear and Cytoplasmic Protein Extraction Kit were from Beyotime; Super ECL Prime was from US Everbright®Inc. The mRNA extraction kit was from Bioteke. Protein A Magnetic Beads and the Muse Count & Viability Assay Kit were purchased from Millipore. Anti-Flag M2 Magnetic Beads was from Sigma-Aldrich. Antibodies for phospho- and total-AMPK, acetyl- and total-p53, SIRT1, Cleaved- and total-Caspase-3, FOXO1, PTEN, β-actin, and acetylated-Lysine were from Cell Signaling Technology; antibodies for P21 was from Abcam; antibodies for Prdx2 was from Santa Cruz Biotechnology.

NTM cells were grown to 80% confluency in 8- or 12-well Acea electric plates containing copper electrodes. The transfection mix contained 1nM anti-K-cadherin, anti-OB-cadherin, or nontargeting siRNAs (OnTarget siRNA; GE Dharmacon, Lafayette, CO, USA) along with 0.83 μl Attractene (Qiagen) per well for Acea plates and 5 μl Attractene per well for regular 12-well plates. Attractene and siRNA were incubated together in a transfection mix in OptiMEM (Thermo Fisher Scientific) for 20 minutes. Trypsinized NTM cells were resuspended in OptiMEM containing 5% FBS and 1% glutamine at a density of 2 × 105/ml and 200 uL of transfection mix was added. Eighteen to 24 hours after transfection, culture medium was changed to Dulbecco's modified Eagle medium (DMEM) low glucose containing 10% FBS, 1% glutamine, and 1% penicillin/streptomycin. NTM cells in 12-well plates were harvested 72 hours after treatment for WB. NTM cells cultured in 8-well Acea plates were placed on the Acea iCelligence system for continuous cell impedance measurements.

To assess the effect of silencing HDAC3 on P0 enhancer-promoter activity, NSΔRafER cells were cultured in 6-well plates until 80% confluence was reached. Cells were transfected with 300ng pGL3-P0-Int-Pro luciferase vector (firefly), 5ng of Renilla and 5nM of scrambled (QIAGEN), HDAC3 #1 or #2 siRNA (Dharmacon) with Attractene (Invitrogen). Complexes were added to the cells and incubated for 2h at 37°C. Cells were lysed 48h following transfection with a passive lysis buffer (Promega). Samples were processed using the dual luciferase reporter assay system (Promega) according to the manufacturer’s instructions.