Quanti luc reagent

THP1-HMGB1-Lucia cells stably express in the cytoplasm a 46.5 kDa fusion protein, HMGB1: Lucia, in which the C-terminus of HMGB1 is fused to the Lucia luciferase. THP1-HMGB1-Lucia cells were used to monitor HMGB1 release. Briefly, THP1-HMGB1-Lucia cells were cultured in 200 μL X vivo media (∼200,000 cells) per well of a round-bottom clear culture 96-well plate (Corning 3799). Cells were treated in different concentrations of beQS-21 or ccQS-21 with or without 0.1 μg/mL LPS for 5 h. Supernatant (20 μL) were collected into flat-bottom white assay 96-well plates (Corning Costa 3922). After adding 50 μL of Quanti-Luc reagent (Invivogen, France), luminescence was detected using an EnVision Multimode plate reader (PerkinElmer, US).

Cells were purchased from InvivoGen. Luciferase reporter signals were measured using Quanti-Luc reagent (InvivoGen) according to manufacturer protocol prior to luminometry.

HEK 293 cells in an opaque white 96 well plate were transfected in triplicate with 50 ng control firefly luciferase plasmid (Promega, USA) and 50 ng of pCpGfree-Lucia constructs, using Lipofectamine 3000 (Invitrogen, USA) according to manufacturer’s instruction. The pCpGfree-Lucia constructs used were either the methylated or mock methylated plasmids containing either region 1 or region 2, as well as a vector containing no insert, and a DNA-free control. After 48 h, the luciferase activity was measured after simultaneously treating an aliquot of the supernatant media with QUANTI-Luc reagent (InvivoGen, USA) to measure Lucia luciferase activity, and treating the adherent cells with Dual-Glo Luciferase reagent (Promega, USA) to measure firefly luciferase activity, and immediately reading the resulting luminescence with a Synergy H1 microplate reader (BioTek, USA) with a gain of 100 units. The reads were background corrected to the DNA-free control, then controlled for transfection efficiency by reporting the ratio of Lucia/firefly luciferase luminescence. These ratios were compared to determine the effect of methylation on luciferase activity using a Student’s t test with a statistical significance threshold of α ≤ 0.05 (Fig. 5b).

Cloned vectors were isolated by Qiagen QIAprep Spin Miniprep kit (Qiagen). M. SssI CpG methyltransferase (New England Biolabs, Frankfurt, Germany) was used for in vitro methylation according to manufacturer’s instructions. Methylated DNA was then purified using the QIAquick gel extraction kit (Qiagen) and quantified by NanoDrop (Thermo Scientific NanoDrop products, Wilmington, DE). Methylation was confirmed by digestion with the methylation-sensitive restriction enzymes HhaI and HpaII. HEK293 cells grown to confluence on 96-well plates were transfected with the pCpG free-Gpx1 vector using Lipofectamine 2000 (Invitrogen). 24 h after transfection, luciferase activity was measured with the QUANTI-Luc reagent (Invivogen, San Diego, CA) by luminescence detection. Promoter activity was normalized to the total amount of protein measured by a Bradford assay (Biorad, Hercules, CA).

For in vitro methylation studies, constructions were done using the CpG-free and promoterless vector pCpG-free basic-Lucia (Invivogen, San Diego, CA) as the backbone. A 469-bp Sod2 promoter region was amplified on mouse genomic DNA using forward BamHI (5′-ATTGGATCCTTTGCAGCTCACAGCCAGAGCTGGACA-3′) and reverse HindIII (5′-TAAAAGCTTAGCCAGCCACGCCCGCCGCCCCG-3′)-linked primers and subsequently inserted in the backbone using BamHI and HindIII restriction sites. Cloned vectors were isolated by Qiagen QIAprep Spin Miniprep kit (Qiagen). M.SssI CpG methyltransferase (New England Biolabs, Frankfurt, Germany) was used for in vitro methylation according to manufacturer's instructions. Methylated DNA was then purified using the QIAquick gel extraction kit (Qiagen) and quantified by NanoDrop (Thermo Scientific NanoDrop Products, Wilmington, DE). Methylation was confirmed by digestion with the methylation-sensitive restriction enzymes HhaI and HpaII. HEK293 cells grown to confluence on 96-well plates were transfected with the pCpG-free Sod2 vector using Lipofectamine 2000 (Invitrogen). Following 24 h transfection, luciferase activity was measured with the QUANTI-Luc reagent (Invivogen) by luminescence detection.

Jurkat-Lucia NFAT cells are derived from the human T lymphocyte-based Jurkat cell line by the stable integration of a nuclear factor of activated T cells (NFAT)-inducible Lucia reporter construct (catalog number jktl-nfat; InvivoGen). Jurkat cells were cultured in RPMI 1640 medium (catalog number 11875-093; Life Technologies) containing 10% fetal bovine serum (catalog number 26140-079; Life Technologies) and 1% penicillin-streptomycin (Life Technologies). The cells were stimulated with PMA-ionomycin in the absence or presence of AgSAP or heat-inactivated AgSAP. Thirty microliters of the cell supernatant was mixed with 50 μl of Quanti-Luc reagent (InvivoGen), and luminescence was measured using a Synergy Mx plate reader (BioTek).