Safire2 microplate reader

Total protein lysates were obtained from PBMCs, and protein content was determined by Bradford protein assay (Bio-Rad Laboratories, Hercules, California Laboratories, Hercules, CA, USA). The determination of 20S Proteasome content was performed in protein lysates as described previously [6 (link)], using 20S Proteasome ELISA Kit (BML-PW0575, Enzo Life Sciences, Farmingdale, NY, USA). Optical density was read at 450 nm on the Safire II microplate reader (TECAN, Männedorf, Switzerland).

The enzymatic activity of both BACE1 and BACE2 is determined by the enhancement of fluorescence intensity upon enzymatic cleavage of the FRET (fluorescence resonance energy transfer) substrate. The cleavage sequence of the substrate is derived from the literature [27 (link)], and a fluorophor and a quencher dye are attached to the Lys side chain at the termini of the substrate. The human recombinant BACE1 and BACE2 FRET assays were performed in 50 mM Acetate, pH 4.5 / 8% DMSO / 100 μM Genapol / 0.002% Brij-35 using the same FRET substrate in a Costar 96-well black polypropylene plate. In dose-response IC₅₀ assays, 10 concentrations of each compound were made using 1:3 serial dilutions in DMSO and pre-incubated with the enzyme for 60 min at room temperature. Subsequently, the FRET substrate was added to initiate the reaction. After 60 min at room temperature, the reaction was stopped by the addition of un-titrated 0.1 M Tris Base to raise the pH above the enzyme active range. The fluorescence intensity of each well was measured on Safire II microplate reader (Tecan, Switzerland), and the IC₅₀s were calculated by fitting normalized activity data with a 4-parameter non-linear regression equation via Screener software (Genedata AG, Switzerland).

To assess TNFα mRNA expression, RAW 264.7 cells were plated in 6-well plates in alpha-MEM medium supplemented with 10% DCC-FBS, at a density of 300,000 cells per well, maintained in culture for 72 h and then exposed to vehicle (0.3% DMSO) or 3 μΜ test compound for 20 min or 24 h prior to stimulation with 100 ng/ml lipopolysaccharide (Sigma L4391) for 1 h. Extraction of total RNA, reverse transcription (RT) and quantitative PCR (qPCR) analysis of mRNA expression levels were performed as previously described [47 (link)]. Relative gene expression levels were calculated by the comparative Ct method using the formula 2^(-ΔCt). Tumor necrosis factor alpha (TNFα) mRNA levels were normalized to the respective levels of glycerol-3-phosphate dehydrogenase (GAPDH). Test compound effects on the relative number of viable cells were assessed using crystal violet and a Safire II microplate reader (Tecan) as previously described [39 (link)]. The difference in optical density at 550 and 690 nm was taken to measure the actual number of viable cells. The following primers were used:
Mouse TNFα:      FW: 5’-TCTCATTCCTGCTTGTGGCA-3’                            RV: 5’-AGGGTCTGGGCCATAGAACT-3’Mouse GAPDH:  FW: 5’-CATGGC CTTCCGTGTTCCTA-3’                            RV: 5’-CCTGCTTCACCACCTTCTTGAT-3’

DENV1-4 FL NS5 dnI assays were performed as described previously [39 (link)]. Briefly, compounds from 0–20 or -100 μM concentrations are two-fold serially diluted into 384-well black opaque plates (Corning Costar), after which 100 nM DENV FL NS5 protein was added and the plates incubated at RT for 20 min. RNA and ATTO-CTP, ATP, GTP and UTP were then added and the plates incubated for another 120 min. Reactions were stopped with buffer containing 25 nM CIP, re- incubated at RT for 60 min and read on a Tecan Safire II microplate reader. For order-of-addition experiments, DENV4 FL NS5 was incubated for one hour at RT with RNA, ATP, and GTP or RNA, ATP, GTP and ATTO-CTP, followed by exposure to serially diluted compounds for 20 min at RT. The missing components (ATTO-CTP and UTP or UTP alone) were added and the reactions continued for 120 min after which STOP buffer was added as before. All datapoints were performed in duplicate wells. Each compound was tested at least twice.

Carbonyl groups of proteins were detected using the “Protein Carbonyl ELISA Kit” (ab238536; Abcam, Cambridge, UK). Measurements were performed in duplicates, and cell lysate samples were diluted 250-fold to 10 μg/mL protein with PBS before adsorption onto 96-well Protein Binding ELISA Plates. Absorbance was measured using the Safire II microplate reader (TECAN, Männedorf, Switzerland), and the protein carbonyl content in unknown samples was determined using the standard curve derived from the BSA standards.

DNA extraction was performed in collected cells after treatment with the NUCLEOSPIN extraction kit. DNA concentration was determined as described in the Real-Time PCR analysis. The measurement of global DNA methylation status was performed by using the Global DNA methylation kit (ab117128, Abcam, Cambridge, UK), and for each sample, the input for the assay consisted of 100 ng of isolated genomic DNA, in technical duplicates. Absorbance was read at 450 nm using a Safire II microplate reader (TECAN, Männedorf, Switzerland), and Global DNA Methylation was calculated as the percentage of methylated DNA (5-mC) in total DNA through the generation of a standard curve and the relevant formula.