Trireagent

Primary macrophages (10,000) seeded in 100 μL in 96-wells plates were, upon adherence of the cells, stimulated with 100, 1, 0.1 or 0 ng/mL LPS (L4268, Sigma-Aldrich, Saint Louis, MO, USA) for 24 h after which the supernatant was collected and stored at −20 °C for further analysis. The cells were subsequently lysed using TriReagent (#11667165001; Roche Diagnostics, Basel, Switzerland) for mRNA isolation. Cells of 2 wells were pooled in order to obtain sufficient amounts of mRNA for further analysis.

For RNA isolation, myoblasts and myotubes were collected in 500 μL Tri-Reagent (Sigma-Aldrich) and stored at −20°C until processing. For protein isolation, cells were collected in 200 μL of RIPA buffer (Millipore) containing PhosStop phosphatase inhibitor cocktail tablets (Roche) and cOmplete Mini, EDTA-free protease inhibitor cocktail tablets (Roche) on ice. Samples were then incubated on ice for 30 min with vortexing to ensure lysis and then centrifuged for 10 min at 16,000 x g at 4°C. Supernatants were collected and stored at −20°C.
Mice were euthanized and muscles were dissected and snap-frozen in liquid nitrogen and stored at −80°C. Tissue was lyophilized for 8 h and pulverized with ceramic beads on dry ice using a Bertin Technologies Percellys Evolution Tissue Homogenizer (Molnar et al., 2021 (link)). Pulverized tissues were then homogenized on ice with a Polytron PT1200-E in either 500 μL Tri-Reagent or 100 μL RIPA buffer supplemented with protease and phosphatase inhibitor cocktail tablets (Roche). Homogenized tissues in Tri-Reagent were stored at −20°C. Homogenized tissues in RIPA buffer were centrifuged for 10 min at 16,000 x g at 4°C, and the supernatant was stored at −20°C.

RNA was extracted using TRI reagent (Thermo Fisher Scientific) according to manufacturer’s instructions. Colonic biopsies were disrupted in TRI reagent using a MagNA Lyser (Roche, Basel, Switzerland) and SiLibeads (Sigmund Lindner, Oldham, UK) prior RNA extraction. Reverse transcription was performed using high capacity complementary DNA (cDNA) reverse transcription kit following manufacturer´s instructions (Thermo Fisher Scientific). Random hexamer primers were used for cDNA generation and specific miR primers for miR analysis. Real-time PCR (qPCR) was performed in a ABI 7900HT fast real-time PCR system (Thermo Fisher Scientific) using TaqMan^® universal PCR master mix, No AmpErase^® Uracil N-Glycosylase (UNG). mRNA expression was detected using TaqMan^® gene expression assays and miR expression using TaqMan^® miR assays according to manufacturer’s instructions. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and small nucleolar RNA C/D Box 44 (RNU44) primers were used as normaliser housekeeping genes for mRNA and miR analyses, respectively. All reverse transcription and real-time PCR (RT-qPCR) reagents were purchased from Thermo Fisher Scientific.

For cell panel analysis, total RNA was extracted using TriReagent (Sigma) and RNA samples then purified using the RNeasy kit (Qiagen). For extraction of reporter mRNA from luciferase reporter transfections, total RNA was extracted using TriReagent and DNA digested using 10 units of DNase I (Roche) for 15 min at 37°C before quenching by addition of 5mM EDTA. Samples were purified by extraction with acidic phenol–chloroform (Ambion) and RNA then purified using the RNeasy kit.

For gene expression analysis, mRNA was isolated from kidney homogenates or cultured cells using TriReagent (#11667165001; Roche Diagnostics) according to the manufacturer's recommendations. All mRNA samples were quantified by spectrophotometry and stored at −80°C until further analysis. One microgram of mRNA was treated with RQ1 DNAse (M6101, Promega, Madison, WI, USA) and subsequently converted to cDNA using M‐MLV reverse transcriptase (M1705, Promega, Madison, WI, USA) and random hexamer primers (#SO142, Fisher scientific, Landsmeer, the Netherlands) according to the manufacturer's recommendations. qPCR and subsequent analysis were performed using sensiFAST No‐ROX PCR master mix (GC Biotech) on a Lightcycler 480 machine and corresponding software (Software release 1.5.0 (1.5.0.39), Roche, Almere, the Netherlands). Expression levels were normalized using the average expression levels of β‐actin, GAPDH and TBP. Primer sequences are listed in Table 1.