Phrl cmv

IHSF001 ((E)-ethyl 4-oxo-4-(thiazol-2-ylamino)but-2-enoate) was obtained from AKos Consulting & Solutions GmbH (Klarenbeek, Netherlands). The syntheses of IHSF058-115 were described in Ref. [18 (link)] and those of IHSF133 and compounds 142-144, in the Supplementary Materials. WA was sourced from Santa Cruz Biotechnology (Dallas, TX, USA) and CEL, from Merck (Kenilworth, NJ, USA). All compounds were dissolved in DMSO at 10 mM. phRL-CMV and pCMV-Luc were obtained from Promega (Madison, WI, USA) and Origene (Rockville, MD, USA), respectively. pCMV-β-Gal was previously described [57 (link)]. Adenovirus Ad-CMV-RLUC was obtained from SignaGen Laboratories (Frederick, MD, USA).

HKe3 ER:HRAS V12 cells were transfected with the indicated siRNAs for 48 h in 24-well plates, followed by 24-h transfection with 250 ng of NFKB reporter and 10 ng of phRL-CMV (Promega, E2261), which constitutively expresses the Renilla luciferase reporter. One day before the measurement of luciferase activity 100 nM 4-OHT was added. Finally, the transcription assay was carried out using the Dual-luciferase® reporter assay system (Promega, E1960) following the manufacturer’s protocol.

CMT-93 cells were transfected with 0.7 μg of each constructed plasmid, 5 ng of phRL-CMV (Promega), and 2.1 μl of X-tremeGENE HP DNA Transfection Reagent (Roche Diagnostics GmbH), following the manufacturer’s instructions. Twenty-four hours after transfection, luciferase activity was measured using the dual-luciferase assay kit (Promega), according to the manufacturer’s instructions. Data shown are mean ± SD values of three independent experiments, and are reported as fold-induction to cells transfected with Luc-mTollip_3´-UTR1-2782.
For the miRNA inhibition assay, each miRNA-specific inhibitor or control inhibitor was transduced into CMT-93 cells at the concentration of 5 nM with the reporter plasmid Luc-mTollip_3´-UTR1-2782, using X-tremeGENE siRNA Transfection Reagent (Roche Diagnostics GmbH). After culturing for 24 h, the cells were lysed to measure luciferase activity, as described earlier. Data shown are mean ± SD values of two or three independent experiments and expressed as values relative to the control without inhibitors.

HEKhTLR4 cells stably transfected with human TLR4, CD14 and MD2 cDNA constructs purchased from Invivogen (San Diego, CA, USA) and HEKhTLR2 cells stably transfected with a human TLR2 cDNA construct (a gift from Dr. Evelyn A. Kurt-Jones, University of Massachusetts Medical School, Worcester, MA, USA) were maintained in DMEM supplemented with 10% FCS and G418 (0.5 mg/ml). HEKhTLR4, HEKhTLR2 and HEK293 cells plated in 24-well plates (Falcon) at 1 × 10⁵ cells/well were transfected with 400 ng of NF-κB-driven firefly luciferase plasmid (pNF-κB-Luc) (Clontech, Mountain View, CA, USA) and 10 ng of CMV promoter-driven Renilla luciferase plasmid (phRL-CMV) (Promega, Madison, WI, USA) for 16 h. After transfection, cells were stimulated with 5 μg/ml hMrp8, 100 ng/ml LPS or 100 ng/ml BLP for 6 h. Luciferase activity was determined using the dual-luciferase reporter assay system (Promega). Transfection efficiency was normalized in all experiments with simultaneously measured Renilla luciferase activities.

Human HMGCS2 promoter-inserted pGL4.10 plasmids [3 (link)], human PPARα expression plasmid (hPPARα-pTargeT) [3 (link)], p3A4-pGL3 [6 (link)], and human PXR (hPXR) expression plasmid (hPXR-pTargeT) [6 (link)] were prepared previously. phRL-TK, phRL-CMV, phRL-SV40, pGL4.31, and PGC1α-expressing pFN21A plasmids were purchased from Promega (Madison, WI, USA). PGC1α-LXXLL-pFN11A—the pFN11A-based plasmid with the nuclear receptor-binding LXXLL motif of PGC1α (EAEEPSLLKKLLLAPANTQ)—and the pFN10A plasmid with hPPARα and hPXR cDNA were prepared as previously described (PPARα-pFN10A and PXR-pFN10A) [3 (link),9 (link)]. hPXRΔAF2-pTargeT was produced using a KOD Plus mutagenesis kit (TOYOBO, Otsu, Japan) with specific primer sets for inserting a stop codon between Ala421 and Thr422.

PPRE in the intron 3 of Angptl4 initially cloned into SEAP vector (gift of Sander Kersten, Wageningen University, The Netherlands) was excised out and cloned into pGL3 vector (Promega) downstream of firefly luciferase reporter gene. Raw macrophages were co- transfected with pGL3 construct (400 ng per well) and Renilla luciferase control plasmid (phRL-CMV; Promega, 1.5 ng per well) in 24-well plates using Lipofectamine 2000 (Invitrogen) according to the manufacturer's guide. 24 h after the transfection, cells were treated with or without Ac-LDL or rosiglitazone (a synthetic PPAR-γ agonist) for another 24 h. Luciferase levels were measured using Dual Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized to the corresponding Renilla luciferase activity and plotted as a percentage of the control. Experiments were performed in triplicate and repeated at least three times.