Pgl4.32 luc2p nf κb re hygro

Neuro2a cells were stably transfected with pGL4.32 [luc2p/NF-κB-RE/Hygro] (Promega, Madison) and then transfected with previous plasmids. After 24 or 48 h, cells were lysed with glo lysis buffer (Promega, Madison) and 1 volume of Bright Glo luciferase assay system (Promega, Madison) was added after 5 min. Each sample was duplicated and the experiments were repeated more than 5 times. Luciferase activity was measured with Enspire reading machine in a 96 wells plate.

MPT0G009 and SAHA were synthesized by Dr. Jing-Ping Liou to >98% purity.¹² The non-conjugated primary antibodies used were against HDAC6 or β-actin (Epitomics Inc., Burlingame, CA, USA), HDAC1, HDAC2, HDAC3 or histone H3 (Cell Signaling Technology, Danvers, MA, USA), or acetyl-histone H3 or p21/WAF1/Cip1 (Millipore, Temecula, CA, USA). Fluorescein isothiocyanate (FITC)-conjugated anti-CD51/61 antibodies was from BD Biosciences (San Jose, CA, USA). The labeled secondary antibodies were horseradish peroxidase- or FITC-conjugated anti-mouse or anti-rabbit IgG antibodies (Jackson ImmunoResearch Inc., West Grove, PA, USA). Fluorogenic HDAC1, 2, 3, 4, 6 or 8 assay kits were from BPS Bioscience Corp. (San Diego, CA, USA), colorimetric HDAC activity kits from BioVision Inc. (Milpitas, CA, USA), PGE₂ immunoassay kits from R&D Systems (Minneapolis, MN, USA) and IL-6 ELISA kits from eBioscience Inc. (San Diego, CA, USA). The pGL4.32[luc2P/NF-κB-RE/Hygro] and pGL4.30[luc2P/NFAT-RE/Hygro] plasmids were from Promega Corp. (Madison, HI, USA) and the HDAC1-Flag (plasmid 13820) and HDAC6-Flag (plasmid 13823) plasmids from Addgene Inc. (Cambridge, MA, USA). TurboFect transfection reagent was from Fermentas (Burlington, ON, Canada). Unless otherwise stated, all other chemicals were from Sigma-Aldrich (St. Louis, MO, USA).

To analyze NFκB activity, MCF-7 cells were transiently transfected with pGL4.32[luc2P/NF-κB-RE/Hygro] (Promega, Madison, WI) containing five copies of a NFκB response element and pGL4-hRluc-TK (Renilla, Promega) for 48 h and treated with 10 ng/ml TNFα and 0–40 µM AnAc 24:1n5 for 6 h before performing dual luciferase assay (Promega). Firefly luciferase was normalized by Renilla luciferase. Values are the average of three separate wells in one experiment ± SEM.
RNA isolation, RT-PCR and quantitative real-time PCR (qPCR) was performed essentially as described previously in MCF-7 and MDA-MB-231 cells treated with vehicle control (EtOH) or AnAc 24:1n5 (13.5 and 35 µM, respectively) for 6 h^{6 (link)}. PCR Primers were synthesized by Integrated DNA Technologies (Coralville, IA) and sequences used were are listed in Supplementary Table 9. GAPDH was used as a reference for normalization^{104 (link)}. qPCR was performed in triplicate using ABI Viia 7 (LifeTechnologies). Fold change relative to vehicle-treated, control cells was estimated by the comparative threshold cycle (Ct) method (2^−ΔΔCT)^{105 (link)}.

HEK293 and 293/hTLR4-MD2-CD14 cells (1.25 × 10⁵ cells/well) were plated into 24-well plates (Corning) containing 500 μL culture medium. After incubation for 24 hours at 37°C, the cells were transfected with 25 ng luciferase plasmid DNA, 25 ng Renilla pGL4.74 (hRluc/TK) vector (Promega, Madison, WI, USA) as an internal control, 500 ng plasmid DNA containing five copies of an NF-κB response element that drove the transcription of the luciferase reporter gene (pGL4.32 [luc2P/NF-κB RE/Hygro]; Promega), and/or a tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) plasmid using Lipofectamine® LTX (Life Technologies). TRAF6 plasmid was a gift by Dr. Koji Nakagawa (Hokkaido University). After 24 hours of incubation at 37°C, the cells were treated with 10 ng/mL LPS for six hours, and the reporter gene assay was performed using the Dual Luciferase® reporter assay system (Promega). Luminescence intensity was measured using the GloMax®-Multi Detection System (Promega) according to the manufacturer's instructions. Transcriptional activity was normalized to the Renilla luciferase activity. All experiments were performed in triplicate.

Transient transfection was performed using Effectene (Qiagen; Santa Clara, CA, USA) according to the manufacturer’s protocol. Briefly, 1.0 × 10⁶ cells were seeded in a 60-mm dish a day before transfection and grown to ~70% confluence. The cells were then transfected with 0.1 µg of plasmid DNA—SBE-luc from the SBE Reporter Kit (BPS Bioscience, CA, USA)—or the NF-κB-luc vector pGL4.32 [luc2P/ NF-κB-RE/Hygro] (Promega, Madison, WI, USA) along with 10 nM siRNA against FPR2 (si-FPR2) or control siRNA. The next day, the transfected cells were treated with or without radiation (6 Gy) for 48 h and harvested for protein extraction, real-time-qPCR, or further analysis. All experiments were performed in triplicate, and representative results were reported. For the reporter assay, cells were lysed and assayed for luciferase activity according to the manufacturer’s instructions (Promega, Southampton, UK).

A Jurkat CD137-NFκB-luc stable reporter cell line was generated by stably integrating the CD137 and the NF-κB luciferase reporter genes into Jurkat E6 cells. In short, the [pIRESneo3] vector (Clontech) containing the CD137 gene was transfected, and stable clones expressing CD137 were generated following antibiotic selection. Next, the NF-κB luciferase reporter constructs pGL4.32 [luc2P/NF-κB-RE/Hygro] (Promega) was transfected into the clone with the highest CD137 expression, and stable clones expressing both CD137 and NF-κB luciferase were selected following antibiotic selection. Clones were selected for their capacity to respond to CD137L after initial activation by plate-bound CD3 antibodies (clone OKT-3) and PMA/ionomycin. Jurkat CD137-NFĸB-luc reporter cells were cultured in 9% FBS-HI RPMI 1640, 2 mM L-Glutamine containing 500 µg/mL G418 (Sigma cat. no. G8168) and 400 µg/mL Hygromycin B gold (Invivogen, cat. no. ant-hg-1).