M mlv enzyme

Total RNA was extracted from cells using the RNeasy kit (Qiagen, Courtaboeuf). RNA (500 ng) was reverse transcribed using the M-MLV enzyme (ThermoFisher Scientific). qPCR, for evaluating eASC differentiation, was performed using MSC PCR array (Qiagen). Analysis of cytokine expression was done using horse cytokines and chemokines RT² profiler PCR array (Qiagen). Other qPCR analyzes were done with Taqman gene expression assay (Life Technologies, Courtaboeuf). PCR reactions were carried out on 20 ng of cDNA samples according to supplier’s recommendations on a 7500 FAST real-time PCR system (Applied Biosystems) and analyzed with the dedicated software. All values were normalized to GAPDH housekeeping gene and expressed as relative expression or fold change using the respective formulae 2^−ΔCT or 2^−ΔΔCt.

Total RNA was isolated using TRIzol reagent (Thermo Fisher Scientific), as described by the manufacturer. Reverse transcription was performed with 1 μg of total RNA using random primers and with M-MLV enzyme (Thermo Fisher Scientific). Real-time quantitative PCR was realized with SYBR green Master Mix (Roche), on a Light Cycler 480 instrument (Roche) as previously described (Escobar et al, 2015 (link)). Ribosomal protein S9 (rS9) and GAPDH were used as an internal control. The sequence of the primers used in this study is indicated in Table S2. Results are expressed as N-fold differences in target gene expression relative to the internal control gene and termed “mRNA expression,” were determined as mRNA expression = 2^{−ΔCtsample}, where the ΔCt value of the sample was determined by subtracting the Ct value of the target gene from the Ct value of the average of the internal control genes. Target genes were considered to be non-detectable when the Ct value was above 35.

Table S2 Sequence of the primers used.

Total RNA was isolated from each sample using RNeasy Mini Kit (Qiagen, Courtaboeuf) and the quantity and purity of the total RNA were determined by using a NanoDrop ND-1000 spectrophotometer (NanoDrop ND, Thermo Fisher Scientific). cDNA was synthesized by reverse transcribing 500 ng RNA into cDNA using the M-MLV enzyme (Thermo Fischer Scientific). Quantitative PCR was performed using the SybrGreen PCR Master Mix (Roche) and a LightCycler^® 480 Detection system, following manufacturer’s recommendations. Specific primers for mouse Vimentin, Cadherin-11, N-Cadherin, E-Cadherin, ICAM-1, Integrinβ1, NCAM, MANF, RPS9 and human TYPE 2B COLLAGEN (Col2B), AGGRECAN (AGN), LINK and SOX9 were designed using the Primer3 software (Table 1). Values were expressed as relative mRNA level of specific gene expression as obtained using the 2^–ΔCt method, using the RPS9 expression as housekeeping gene.

Aortic tunica intima and media tunicas were independently cryo-pulverized in liquid nitrogen using a freezer mill (model 6750 SPEX SamplePrep). Total RNA was extracted from the pulverized tissues and vSMCs using TRIzol reagent (Life Technologies) according to the manufacturer’s protocol. Reverse transcription was performed using the MMLV enzyme (Life Technologies), and real-time PCR was conducted in a LightCycler system using a SYBR green detection kit (Roche Applied Science) with specific primers (Table 1).

For the validation of gene expression of selected genes in infected macrophages (saprophyte, culture-attenuated and virulent strains) and non-infected control macrophages, RNA samples were reverse transcribed (1μg of total RNA/sample) using the Moloney Murine Leukemia Virus (MML-V) enzyme (Life Technologies) and Oligo-dT Primers. All primers were designed to span at least one intron, to avoid repeat regions and similarities to other non-specific genomic regions. Mouse genome sequence, available on the University of California, Santa Cruz (UCSC) Genome Browser, was employed for primer design, using the Primer3 program [34 (link)]. PCR was performed using a Stratagene QPCR Systems Mx3005P (Agilent Technologies, Santa Clara, CA, USA) using the QuantiTect SYBR Green PCR kit (Qiagen). Expression levels were determined using standard curves for all genes at each individual run, and the expression of the candidate gene is presented as a ratio to an unregulated endogenous control (β-actin).