Primer express software package v2

Total RNA from cardiac tissue was isolated using TRIZOL (Invitrogen) and SV Total RNA Isolation System (Promega, Madison, WI, USA) according to the manufacturer’s instructions. cDNA was synthesized using 500 ng of RNA through a reverse transcription reaction (ImProm-IITM Reverse Transcriptase, Promega). Real-time PCR quantitative mRNA analyses were performed in a StepOnePlus Real-Time PCR System (Applied Biosystems) using SYBR Green reagents (Invitrogen) for quantification of the amplicons. The standard PCR conditions were as follows: 50°C (2 min), 95°C (10 min); 40 cycles of 94°C (30 s), 58°C (30 s), and 72°C (1 min); followed by a standard denaturation curve. Primers were designed by using the Primer Express software package v2.0 (Applied Biosystems), based on the nucleotide reference sequences available at GenBank database. Platinum SYBR Green qPCR SuperMix UDG with ROX reagent (Invitrogen), 1 mg/mL of each specific primer and a 1:20 dilution of cDNA were used in each reaction. The mean Ct values from duplicate measurements were used to calculate the expression of the target gene, with normalization to internal controls (GAPDH, β-actin, and HPRT).

For BM lysates and isolated pMo and inflammatory Ly6C^high monocytes, the total RNA was extracted using the RNAlater^® (Sigma-Aldrich) and PureLink™ RNA Mini Kit (Invitrogen™) according to the manufacturer instructions. For liver homogenates, the total RNA was extracted using the TRIzol^® and the SV Total RNA Isolation System Kit (Promega^®) according to the manufacturer instruction. Complementary DNA was synthesized with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). SYBR Green Mix–based real-time quantitative PCR assays were performed using the StepOnePlus Real-Time PCR System (Applied Biosystems). The mean cycle threshold (Ct) values of triplicate measurements were used to calculate the expression of the target genes, which were normalized to the housekeeping gene Gapdh to liver and BM cells, and analyzed with the 2^-ΔΔCt method. All primers (Supplementary Table 1) were designed using the Primer Express software package v2.0 (Applied Biosystems), based on the nucleotide reference sequences available at GenBank database.