Tissuelyzer lt

Nasopharyngeal swabs preserved in PrimeStore nucleic acid preservation medium (Longhorn Vaccines and Diagnostics, San Antonio, TX, USA), transported on ice and frozen at –80°C for batch testing. Swabs underwent mechanical lysis on a Tissuelyzer LT (Qiagen, Hilden, Germany) followed by total nucleic acid extraction (QIAsymphony Virus/Bacteria Mini Kit, Qiagen, Hilden, Germany). Quantitative, multiplex, real-time PCR (qPCR) with FTDResp33 (Fast-Track Diagnostics, Esch-sur-Alzet, Luxembourg) identified potential respiratory pathogens including sHCoV (-NL63, −229E, -OC43, -HKU1). Standard curves were derived using standards supplied by the manufacturer.

Protein lysates from adipose tissues were extracted using Qiagen TissueLyzer LT and NP-40 lysis buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl, EDTA 5mM, 1% NP-40 and cOmplete protease inhibitors (Roche). The tissue lysates were immunoprecipitated using the antibody against SERCA2 (Thermo Fisher, 2A7-A1, 1:200). Mouse IgG was used to confirm the specificity of immunoprecipitation. The immunoprecipitants were subsequently applied for immunoblot using the SERCA2 antibody. β–actin (Sigma, A3854, 1:10,000) was used as loading control for each sample (input). Rabbit polyclonal PRDM16 antibody (1:1,000) was developed previously^{12 (link)}. UCP1 antibodies for mouse (Abcam, ab10983, 1:1000) and human (Sigma, U6382, 1:1,000) were used to detect UCP1 proteins. Mitochondrial proteins were detected using MitoProfile® total OXPHOS rodent antibody cocktail (Abcam, ab110413, 1:1000).

RNA was isolated using the standard Trizol protocol, with a few changes. Briefly, frozen heart sections were lysed in Trizol using the TissueLyzer LT (Qiagen) followed by chloroform addition. The samples were centrifuged for 15 min at 12,000 × g at 4 °C. The upper aqueous phase was transferred to a new tube and 500 μL of isopropanol was added to each sample, followed by another incubation for 2 h at −20 °C. Samples were centrifuged for 10 min at 12,000 × g at 4 °C. The supernatants were discarded, and the pellets were resuspended in 1 mL of 75% ethanol. The tubes were vortexed and centrifuged for 5 min at 7500 × g at 4 °C, and the supernatants were discarded. The pellets were air-dried for 10 min and resuspended in 50 μL of RNAse-free water and stored at −80 °C until further analysis. For cDNA synthesis, 1 μg of RNA was used for each 20 μL reaction following the iScript Reverse Transcription Supermix protocol (Bio-Rad). ASPN (5′-TCCAGCAAAGTTGGTGGTAG-3′, 5′-CCTCTTGAGAACAACGGGATAG-3′) expression analysis by quantitative real-time PCR reaction (qPCR) was performed in the CFX96^™ Real-Time System (Bio-Rad) using 50 ng of cDNA and following the iTaq Universal Sybr Green Supermix (Bio-Rad). Gapdh was used as endogenous control. Expression results were analyzed using the 2-del-taCt formula and represented in graphs as fold change.