Dataassist software

Total RNA isolation was carried out by using Gen Elute Mammalian Genomic Total RNA Kit (Sigma) from untreated and quercetin-treated HeLa cells (25 and 50 µM for 48 h) and cDNA was synthesized (ABI RT-PCR Kit). The synthesized cDNA was then used as a template for TaqMan® Human Apoptosis Array (Thermo Fisher), which has a range of different apoptosis regulators from both intrinsic and extrinsic pathways. A TaqMan-based custom array was designed consisting of several tumor suppressor genes and regulatory genes from various signal transduction pathways. PCR array was run on QuantStudio3 and analyzed by the ΔΔC_T method using DataAssist™ software from Thermo Fisher. The data were normalized using 18s rRNA expression (apoptosis array) and global normalization (custom array). RQ indicates the fold change in gene expression against untreated control after normalization with the selected endogenous gene.

To study differential gene expression, RNA was extracted from lung tissues using Trizol, subjected to cDNA synthesis (High Capacity cDNA Reverse Transcription Kit, Thermo Fisher Scientific) and qPCR using a custom Taqman qRT-PCR array (Thermo Fisher Scientific) of 30 genes known to be activated in response to virus infection¹³ , as well as two housekeeping genes (Supplementary Table 2). Data collected were analyzed using the Quant Studio Design and Analysis (version 1.5.1) and Data Assist software (version 3.01, Thermo Fisher Scientific). Pathway, Gene Ontology, and transcription factor target enrichment analysis was performed using Gene Set Enrichment Analysis (Molecular Signatures Database (MSigDB), Broad Institute). Principal component analysis, correlation matrices, and unsupervised hierarchical clustering (Euclidean distance) were performed using XLSTAT and visualized using MORPHEUS (https://software.broadinstitute.org/morpheus)¹⁷ .

Total RNA from cells was isolated by following the TRIzol method. The concentration of RNA and their structural integrity were confirmed by using a Nanodrop 2000 UV–Vis spectrophotometer (Thermo Scientific). Only RNA with the ratios from 1.9- 2.0 of absorbance at 260/280 nm has been used. The isolated mRNA was reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (4368814) according to the protocol. Quantitative reverse transcription-PCR (q RT-PCR) was performed by using PowerUp SYBR green master mix (ABI- A25741) on a 7500 Fast Real-Time PCR system (Applied Biosystem, Thermo Scientific). Quantification was performed with the ΔΔ Ct method with β-actin serving as a reference gene, and the RT-PCR results were analyzed by DataAssist software (Thermo Scientific). The oligonucleotide primers are listed in Table S1. All primers were analyzed with positive controls by performing melting profiles following q RT-PCR, and product sizes were checked by 2.2% agarose gel electrophoresis. PCR conditions were as follows: 40 cycles of 15 seconds at 95°C, 15 seconds at annealing temperature (60°C for all other genes), and 15 seconds at 72°C. Specimens were assayed in duplicate for at least three independent experiments as indicated.

Total RNA was extracted from peritoneal cells with the Illustra™ RNAspin Mini Kit (GE Healthcare Lifesciences, Buckinghamshire, UK). Quantification and purity of the samples were measured in a NanoVue spectrophotometer (GE Healthcare Lifesciences), and integrity was determined in the Bioanalyzer 2100 instrument with the RNA 6000 Nano Kit (Agilent, Waldbronn, Germany). Samples were used if their RIN (RNA Integrity Number) was at least 7.0. RNA (250 ng) was reverse transcribed with GoScript Reverse Transcriptase (Promega, Madison, USA) and oligo dT₁₈ primers. qRT-PCR reactions were carried out in a StepOnePlus real-time detector using SYBR FAST mastermix (Life Technologies, Foster City, USA) in a final volume of 10 μL. The expression levels of several reference genes (Rps29, Tbp, Gapdh, Ppia, and B2m) were analyzed for stability with the geNorm VBA applet [18 (link)], and the combination of Ppia and B2m was determined as the most stable. Primer pair sequences are described in Table 1 and were designed with Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/), with the exception of Mx1, which was obtained from PrimerBank (https://pga.mgh.harvard.edu/primerbank/; PrimerBank ID 6996929c1).
Normalized (2^−ΔCt) gene expression [19 (link)] was calculated with DataAssist Software (Thermo Fisher Scientific, Foster City, USA) using the mean of Ppia and B2m Ct values as reference.

Cells were lysed in TRIzol reagent (Thermo Fisher Scientific), and RNA was extracted and reverse-transcribed into cDNA using SuperScript II Reverse Transcriptase (Life Technologies, Thermo Fisher Scientific) as recommended by the manufacturers and previously described (60 (link)). Gene expression analysis was performed using predesigned primers and probes (Thermo Fisher Scientific). All TaqMan analysis was performed using an Applied Biosystems (Thermo Fisher Scientific) ViiA 7 instrument, and fold change values were calculated using Data Assist software (Thermo Fisher Scientific). All human and mouse data were normalized to RNA18S5 or GAPDH expression, respectively.