Pgc 1α

Soleus muscles were isolated from wild type, NSE/IL-6, MCK/PGC-1α, and MCK/PGC-1α:IL-6 mice of 6 months of age. For RNA extraction, muscles were frozen in liquid nitrogen, powdered and homogenized in TRI Reagent (Sigma-Aldrich) by using Tissue Lyser (Quiagen, Hilden, Germany). The reverse transcription was performed by using QuantiTect Reverse Transcription Kit (Quiagen) according to the manufacturer’s instructions. Real time PCR analysis was performed on ABI PRISM 7500 SDS (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) by using specific TaqMan assays (Atrogin1, MURF-1, PGC-1α, MEF2c, and AchR-γ; Applied Biosystems). Relative quantification was performed using and β-actin (Applied Biosystems, USA) as endogenous control. Data were analyzed using the 2-DDCt method and reported as mean fold change in gene expression relative to wild type.

For RNA isolation, CM cells were further purified away from remaining NM cells by gentle resuspension and centrifugation in ice-cold PBS (3×1 min) at 500 rpm at 4°C, then resuspended in 1 ml TRI-reagent (Ambion, UK). The remaining NM-cell-containing supernatant from the initial CM settling was further cleared of CM cells by sequential centrifugation (4×1 min) at 500 rpm at 4°C until no CM pellet was visible. The remaining supernatant was centrifuged at 1500 rpm for 10 min to pellet the NM population, which was resuspended in TRI-reagent for RNA isolation following the manufacturer's instructions. For whole heart RNA, ten 30-µm cryo-sections were cut using a Leica cryostat, homogenised in 1 ml TRI-reagent, then processed as before. All RNA samples were treated with TURBO DNase (Ambion, UK) and reverse transcribed into cDNA with Superscript III (Invitrogen, UK) using random hexamers (Sigma). For qPCR analysis, cDNAs were added to reactions containing Taqman PCR master mix, gene-specific primer-probe sets for Col1α1, Tgfβ, Vegfα, Tnc, IL-6, IL-1β, Cxcl-2, Anf, Serca2α, Ryr2, Pgc1α and Gapdh (Applied Biosystems), and amplified using an ABI-7500-PCR system. All samples were normalised to Gapdh. Relative expression was calculated as 2^−ΔCT×100 and presented as % Gapdh.

Heart tissues used for RNA isolation were rapidly excised and frozen in liquid nitrogen. RNA was extracted from heart samples by using TRIzol reagent (Invitrogen, USA) method following manufactory's protocol. The integrity of total RNA was assessed using Nano Drop 2000 Spectrophotometer (Thermo, USA). Total RNA was reverse transcribed by Invitrogen 2-step RT kits Superscript II first strand synthesis kit (Invitrogen, USA) according to the manufacturer's instructions.
The cDNA was 1:20 diluted and used for real-time PCR. Platinum SYBR Green qPCR Super Mix-UDG with ROX kits (Invitrogen, USA) were used to measure gene expression using specific primer sets: pdk4, glut4, ampk, pgc-1α, and gapdh (Invitrogen, USA) (Table 1). Dissociation curves were run following Real-Time PCR reactions to ensure the detection of the desired amplicon and exclude the presence of contaminating products. All reactions were performed in the ABI Prism 7000 Sequence Detection System (Applied Biosystems, USA). Gene expression was normalized to gapdh and the data were analyzed using comparative 2^∧−ΔΔCt method.

Protein samples were prepared from liver homogenates in Laemmli sample buffer, run on SDS-polyacrylamide gels (4-15% TGX stain-free gel, Bio-Rad), and transferred to the polyvinylidene difluoride (PVDF) membrane. The membranes were blocked, incubated with primary antibodies overnight at 4 °C, followed by secondary antibodies, and developed using the chemiluminescence imaging system (Bio-Rad). Following primary antibodies were used: OPA1 (BD Biosciences, 612606; 1:1000); caspase-3 (Cell Signaling, 9662; 1:1000), PARP-1 (Cell Signaling, 9542; 1:1000), β-actin (Sigma, A1978; 1:40000), TOM20 (Proteintech, 11802-1-AP; 1:1000), cytochrome c (BD Biosciences, 556432; 1:5000), elF2α (Cell Signaling, 9722; 1:1000), phospho-elF2α (Cell Signaling, 9721; 1:1000), FGF21 (Proteintech, 26272-1-AP; 1:1000), LC3 A/B (Cell Signaling, 4108; 1:1000), PGC1α (Invitrogen, PA5-38022; 1:500), OMA1 (Santa Cruz, sc-515788; 1:100), and mitochondria total OXPHOS rodent WB cocktail (Abcam, ab110413; 1:1000). JNK (Cell Signaling, 9252; 1:1000), p-JNK-Thr183/Tyr185 (Cell Signaling, 9255; 1:500), MCU (Sigma, HPA016480; 1:1000), NCLX (Proteintech, 21430-1-AP; 1:1000), CypD (Proteintech, 18466-1-AP; 1:1000), MnSOD (BD Biosciences, 611580; 1:1000), GPx1/2 (Santa Cruz Biotechnology, sc-133160; 1:200), and CYP2E1 (Proteintech, 19937-1-AP; 1:300).

Immunostaining was carried out using the following antibodies: lamin A/C (MAB3211; Millipore, Gibbstown, NJ, USA), progerin (Cao et al., 2011b), PGC‐1α (Thermo Scientific, USA), and DAPI (Vector Laboratories, Burlingame, CA, USA) was used to counterstain cell nuclei. Images were acquired with either Zeiss AX10 microscope equipped with a SPOT PURSUIT camera or Zeiss, USA LSM 710 confocal microscope. Fluorescence intensity was analyzed with ImageJ or a custom program (Driscoll et al., 2012).