Nanodrop spectrometer

Quantitative real-time PCR (qPCR) analysis of the gene expression of β-catenin, cyclin D1, cMYC, Wnt3a, and Rac1 was performed in mouse ileum tissue stored in RNAlater solution (Qiagen), an RNA stabilizer, at −80°C. Total RNA was extracted by an RNA isolation system (Promega) according to the manufacturer’s protocol. The RNA was quantified by a NanoDrop spectrometer (Thermo Fisher Scientific), and RNA quality was determined by examining the 260/280 ratio > 1.8. A total of 1 μg RNA was then reverse transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems) according to the manufacturer’s protocol. qPCR was performed using SYBR Green PCR Master Mix (Applied Biosystems), as described in the manufacturer’s instructions. The sequences of the primers are listed in Table 1. To compare gene expression under different conditions, the expression under each condition (normalized to GAPDH, the endogenous control) was quantified relative to the control condition. For β-catenin, cyclin D1, cMYC, Wnt3a, and Rac1, qPCR amplification was performed in a CFX Connect system (Bio-Rad) under the following conditions: 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. The relative expression levels of the genes were calculated using the threshold cycle (2^−ΔΔCT) method (Livak and Schmittgen, 2001 (link)).

Transfected HeLa cells after EV harvest, and Huh‐7 cells after incubation with EVs carrying cel‐miR‐39, were lysed in TRIreagent (Sigma‐Aldrich) and small RNA isolation was performed using miRNeasy Mini kit (QIAgen) according to manufacturer's instructions. RNA concentration and quality were assessed using Nanodrop spectrometer (ND‐ONE‐W; Thermo Fisher Scientific).

Total RNA was extracted from frozen mouse ventricles using TRI Reagent (Sigma-Aldrich, St Louis, MO) and quantitated with a Nanodrop™ Spectrometer (Thermo Scientific, Waltham, MA). To generate protein lysates, frozen mouse ventricles or atria were homogenized in lysis buffer (10% glycerol, 137 mM NaCl, 20 mM Tris-HCl (pH 7.4), 20 mM NaF, 10 mM EDTA, 1 mM EGTA, 1 mM sodium pyrophosphate, 1 mM vanadate, 1 mM PMSF, 4 μg/mL pepstatin, 4 μg/mL aprotinin, 4 μg/mL leupeptin) and the Bradford method (Biorad) was used to measure total protein concentration.

Total RNA was purified from MDMs derived from an additional donor and stimulated with LPS (200 ng/ml) for 6 h. Unstimulated cells were collected at the same time point for use as control. RNA was quantified and assessed for purity using a nanodrop spectrometer (Thermo Fisher). Equal amounts of RNA (500 ng) were reverse-transcribed using iScript Reverse Transcription Supermix (Biorad, 1708841) followed by qPCR using iQ SYBR Green Supermix reagent (Biorad, 1725122) with the following parameters: 50 °C for 2 min, 95 °C for 2 min followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 45 s. Oligos used in RT-qPCR analysis were designed using Primer3 Input version 0.4.0 (https://bioinfo.ut.ee/primer3-0.4.0/). Primer sequences are provided in Table S9. Gene expression levels were normalized to Hprt as a housekeeping gene.

Prepare RNase-free reagents and consumables in advance. TRIzol reagent is used to lyse the cell sample to be tested, and then extract total RNA. The absorbance of the extracted total RNA at 280 and 260 nm was detected by nanodrop spectrometer (Thermo Scientific) to evaluate its purity and concentration. Use the first-strand cDNA synthesis super-Mix kit to reverse transcribe qualified total RNA samples into cDNA. Real time quantitative PCR was performed in the light cycle 96 real-time PCR system (Roche) using aceq qPCR SYBR Green master mix. The mRNA expression level of IL6 was detected using a forward primer: 5′-ACA GCC ACT CAC CTC TTC AG-3′ and a reverse primer: 5′-CCA TCT TTT TCA GCC ATC TTT-3′, GAPDH was used as an internal control and was detected using forward primer: 5′-TGG TAT CGT GGA AGG ACT CA-3′ and reverse primer: 5′-CAG TAG AGG CAG GGA TGA TG-3’.

Total RNA from cells was extracted using a RiboEx (Geneall, Korea) and a Qiagen RNA isolation kit (Qiagen, Valencia, CA, USA) according to manufacturer's instruction. The total RNA concentration was measured by Nanodrop spectrometer (Thermoscientific). Generation of first strand cDNA from total RNA and amplification of this cDNA strand were reacted together using Primer ScriptTM one step RT-PCR kit Ver2 (TAKRA, Japan) and PCR machine (Bio-rad, Richmond, CA, USA) according to manufacturer's instructions. Differently expressed mRNA was separated by 1.2 % agarose gel electrophoresis. Specific primer sequences are as follows:
MMP-1 fwd, 5′-GATGGGAGGCAAGTTGAAA A-3′; MMP-1 rev, 5′-CTGGTTGAAAAGCATGAGCA-3′; CXCL1 fwd, 5′-GAAAGCTTGCCTCAATCCTG-3′; CXCL1 rev, 5′-CCCTGCCTTCACAATGATCT-3′; PTGS2 fwd, 5′-TGCTGTGGAGCTGTATCCTG-3′; PTGS2 rev, 5′-GACTCCTTTCTCCGCAACAG-3′;
TNC fwd, 5′-GTCACCGTGTCAACCTGATG-3′; TNC rev,5′-TCCCAGAGCCACCTAAGAGA-3′;
ACTB fwd, 5′-GCTCGTCGTCGACAACGGCTC-3′; ACTB rev, 5′-CAAACATGATCTGGGTCATCTTCTC-3′.

Total RNA of CVP were extracted using a total RNA purification kit (Norgen Biotek, Canada). Briefly, the nitrogen-frozen CVP were homogenized in the lysing buffer, and after a selection on columns, RNA previously treated with a DNase (RNase-free DNase I kit, Norgen Biotek, Canada) were assayed using a nanodrop spectrometer (Thermo Fischer Scientific). RT-PCR were performed using the following primers (Life Technologies, Thermo-Fisher, France): Arntl/Bmal1, Mm00500223m1; CLOCK, Mm00455950m1; Per2, Mm00478099m1; Cry2, Mm01331539m1; NR1D1/RevErbα, Mm00520708m1; NR1D2/RevErbβ, Mm01310356g1; CD36, Mm00432403m1; GPR120/FFAR4, Mm00725193m1; PLCβ2, Mm01338057m1; Tas1R3, Mm00473459g1.