Metabolic reprogramming of fibro/adipogenic progenitors facilitates muscle regeneration

Mouse models

C57BL/6J (RRID:IMSR_JAX:000664) and C57BL10ScSn-Dmd^mdx/J mice (RRID:IMSR_JAX:001801) (referred to as wt and mdx, respectively) were purchased from the Jackson Laboratories. In our study, sexes were equally balanced between genotypes. Young (45- and 49-d-old) wt and mdx mice were used in this work. Mice were bred and maintained according to the standard facility procedures. All experimental studies were conducted according to the rules of good animal experimentation I.A.C.U.C. n ̊432 of March 12, 2006, and under ethical approval released on December 11, 2012, from the Italian Ministry of Health, protocol #20/01-D.

Murine primary cells

FAPs and MuSCs were isolated from the hind limbs of male and female wt and mdx mice. Freshly sorted FAPs were resuspended in FAPs-GM consisting of DMEM GlutaMAX (25 mM Glc) supplemented with 20% FBS, 10 mM Hepes, 1 mM sodium pyruvate, and 100 U/ml penicillin/streptomycin (P/S). Freshly purified MuSCs growth medium (MuSCs-GM) consisting of DMEM GlutaMAX (25 mM Glc) supplemented with 20% FBS, 10% donor horse serum, 2% chicken embryo extract, 10 mM Hepes, 1 mM sodium pyruvate, and 100 U/ml P/S. The cells were cultivated at 37°C in 5% CO₂.

Diet treatment

At the weaning day, wt and mdx littermate were individually housed and randomly assigned to the LFD or HFD group with free access to food and water. In both groups, sexes were equally balanced between genotypes. Mice were fed with LFD (containing 11 kcal% fat, Research Diet) and HFD (containing 58 kcal% fat, Research Diet D12331) for 4 wk (28 d). Body weight, food, and water intake were monitored every 2 d. Although LFD and HFD differed in their fat content, both diets are isocaloric. Brain, spleen, liver, adipose tissue depots, and hearth were weighted and explanted from each mouse, snap-frozen in liquid nitrogen, and stored at −80°C for further investigations.

Isolation of MuSCs and FAPs from skeletal muscles

Hind limbs were surgically removed and then minced in HBSS (GIBCO) supplemented with 100 U/ml P/S (Roche) and 0.2% BSA (AppliChem). For each mouse, the homogeneous muscle tissue preparation was enzymatically digested in 2 μg/μl collagenase A, 2.4 U/ml dispase II, and 10 μg/ml DNase I (Roche) in Dulbecco’s phosphate-buffered saline (BioWest) w/calcium and magnesium. The enzymatic digestion was performed for 1 h at 37°C with gentle shaking. The digested tissues underwent consecutive filtration through 100, 70, and 40 μm cell strainers (Corning). Before each filtration step, the cells were centrifuged at 700g for 10 min at 4°C and then resuspended in fresh HBSS. Red blood cells were lysed in RBC lysis buffer (Santa Cruz). Freshly isolated muscle mononuclear cells were resuspended in Magnetic beads buffer (0.5% BSA and 2 mM EDTA in 1× PBS) and filtered through a 30-μm Pre-Separation Filter (Miltenyi) to remove large particles from the single cell suspension. The whole cell suspension underwent subsequential incubations with the microbead-conjugated antibodies used for the magnetic sorting. CD45⁺ immune cells and CD31⁺ endothelial cells were collected through the consecutive incubation with the anti-CD45 and anti-CD31 antibodies (Miltenyi). Lineage negative (Lin-) cells were incubated with anti-α7–integrin antibodies (Miltenyi) and MuSCs selected as Lin-/α7-int+ cells. Last, Lin-/α7-int+ cells were incubated with anti-Sca-1 antibodies (Miltenyi) and FAPs selected as Lin-/α7-int+/Sca-1+. The sorting procedures and the labelling procedures with the microbead-conjugated antibodies were performed according to the manufacturer’s instructions.

Lentiviral transduction and FAP transplantation

FAPs were isolated from 49-d-old mdx mice fed with LFD or HFD (n = 3) and transduced through spinoculation (Judson et al, 2018) with one MOI of pLenti CMV GFP Hygro (Addgene). Briefly, 1.0 × 10⁵ cells were plated in serum-free medium (DMEM GlutaMax) in six-well plate and one MOI of virus was administered dropwise. The plate was centrifuged using a swinger rotor adapter for 5 min at 3,200g at 25°C, then for 1 h at 2,500g at 25°C. Virus-containing medium was discarded and the cells were washed twice with serum-free medium and scraped using a soft-gummy blade cell scraper. The cells were separated through centrifugation and resuspended in 100 μl 1× PBS w/Ca²⁺ and Mg²⁺. The cells were inoculated in TA muscles of anesthetized mice. TA muscles were collected after 15 d.

FAP culture and differentiation

Freshly sorted FAPs were resuspended in FAPs-GM consisting of high-glucose (25 mM) DMEM GlutaMAX supplemented with 20% FBS, 10 mM Hepes, 1 mM sodium pyruvate, and 100 U/ml P/S and plated in 96-well plates at a cell density of 3.0 × 10⁴ cells/cm². After 3/4 d, the FAP-GM was fully refreshed and cells cultured for two additional days before the induction of the adipogenic differentiation. The adipogenic differentiation was induced incubating FAPs with the adipocyte differentiation medium (ADM: FAPs-GM supplemented with 1 μg/ml insulin, 0.5 mM 3-isobutyl-1-methylxanthine, and 1 μM dexamethasone) for 3 d followed by two additional days in adipocyte maintenance medium (AMM: FAP-GM supplemented with 1 μg/ml insulin). Unstimulated cells were maintained in fresh FAP-GM. For allowing spontaneous FAP growth and adipogenesis, the cells were plated in 96-well plates at a density of 7.5 × 10³ cells/cm² in FAPs-GM. The medium was refreshed every 2 d for 20 d. Metabolic perturbations were performed during the differentiation period by supplementing ADM and AMM with 250 μM of 2-DG or by replacing Glu with Gal. Palmitate and oleate were dissolved in ethanol at 200 mM. Each fatty acid was diluted separately at 3 mM in a solution of 10% BSA in PBS 1× w/Ca²⁺, Mg²⁺, and incubated overnight at 37°C in gentle shaking. Solution were filtered through a syringe filter of 0.22 μm. Separate stock solution were aliquoted and stored at −20°C. Freshly thawed aliquots were used for treatments. Pharmacological blockade of GSK3β was performed at the onset of the adipogenic differentiation, by supplementing ADM with 20 nM LY2090314. Further experiments involving the use of LY2090314 were performed using in vitro subcultured FAPs. In this case, FAPs were grown in Cyto-Grow (Resnova) by plating 2.5 × 10³ cells/cm². The cells were not passaged more than once and routinely checked for their multipotency. Near-to-confluent cells were dethatched and cultured as described above in FAP-GM.

MuSC culture and differentiation

To reduce fibroblasts contamination, freshly sorted wt and mdx MuSCs were pre-plated for 2 h in pre-warmed MuSC Growth Medium (MuSCs-GM) consisting of high-glucose (25 mM) DMEM GlutaMAX supplemented with 20% FBS, 10% donor horse serum, 2% chicken embryo extract, 10 mM Hepes, 1 mM sodium pyruvate, and 100 U/ml P/S. Pre-plated MuSCs were seeded in MuSC-GM at a cell density of 1.5 × 10⁴ cells/cm² in Matrigel-coated 96-well plates. After 3 d, MuSC-GM was fully refreshed and cells cultured for three additional days allowing spontaneous myotube formation. Metabolic perturbations were carried out in the last 3 d of MuSC differentiation by incubating cells with the MuSC-GM supplemented with 250 μM of 2-DG or by replacing Glu with Gal.

Follistatin (FST) ELISA assay

The Follistatin concentration in FAP-derived culture supernatants was assessed using Custom Mouse Follistatin ELISA Kit (RayBiotech) according to the manufacturer’s instructions. FST standard curve was prepared through serial dilution. FAP CM were diluted 1:4 with Assay Diluent. 100 μl of standard/samples were loaded in duplicate. Colorimetric assay was performed at 450 nm.

FAP-derived CM

Freshly sorted FAPs from mice fed with LFD and HFD were plated in FAPs-GM at a density of 3.0 × 10⁴ cells/cm². After 4 d of culture, CM were recovered, centrifuged at 3,000g to remove cells, and stored at −80°C before use. The control CM was prepared by incubating empty wells with the same medium. CM treatments were performed on 2-d cultured mdx MuSCs. Briefly, MuSC-GM was removed and replaced by FAP-derived CMs. Cells were fixed 3 d later.

Proteome sample preparation

Cells were lysed in sodium deoxycholate (SDC) lysis buffer containing 4% (wt/vol) SDC, 100 mM Tris–HCl (pH 8.5). Proteome preparation was done using the in StageTip (iST) method (Kulak et al, 2014). Samples were separated by HPLC in a single run (without pre-fractionations) and analyzed by MS.

LC-MS/MS measurements

The peptides were separated on a reverse-phase column (50 cm, packed in-house with 1.9-μm C18-Reprosil-AQ Pur reversed-phase beads) (Dr Maisch GmbH) over 120 min (single-run proteome analysis). Eluting peptides were electrosprayed and analyzed by tandem MS on a Q Exactive HF (Thermo Fischer Scientific) using higher-energy collisional dissociation (HCD)-based fragmentation, which was set to alternate between a full scan followed by up to five fragmentation scans.

Proteome data processing

Raw MS data were analyzed in the MaxQuant environment (Cox & Mann, 2008), version 1.5.1.6, using the Andromeda engine for database search. MS/MS spectra were matched against the Mus Musculus UniProt FASTA database (September 2014), with an false discovery rate (FDR) of <1% at the level of proteins, peptides, and modifications. Enzyme specificity was set to trypsin, allowing cleavage of N-terminal to proline and between aspartic acid and proline. The search included cysteine carbamidomethylation as a fixed modification, and N-terminal protein acetylation and oxidation of methionine. Label-free proteome analysis was performed in MaxQuant. Where possible, the identity of peptides present but not sequenced in a given run was obtained by transferring identifications across LC-MS runs (“match between runs”). Up to three missed cleavages were allowed for protease digestion and peptides had to be fully tryptic.

Proteome bioinformatics data analysis

Bioinformatics analysis was performed in the Perseus software environment (Tyanova et al, 2016). Statistical analysis of proteome was performed on logarithmized intensities for those values that were found to be quantified in any experimental condition. To identify significantly modulated proteins, missing values (proteins not identified or not quantified) were imputed by using a normal distribution. Then, we performed a t test with a P-value cutoff of 0.05 and S0 = 0.1. Categorical annotation was added in Perseus in the form of gene ontology (GO) biological process (GOBP), molecular function (GOMF), and cellular component (GOCC), and KEGG pathways and Keywords (extracted from Uniprot). We performed a 2D annotation enrichment analyses to identify statistically significant GO-terms, KEGG pathways, and Keywords enriched in mdx HFD FAPs (Cox & Mann, 2008). Multiple hypothesis testing is controlled by using a Benjamini-Hochberg FDR threshold of 0.07. Then for each term, the corresponding P-value and score are assigned. Whereas a score near 1 indicates a positive enrichment, a score near −1 means a negative enrichment of the category. The complete proteome measurements for FAPs and MuSCs are collected in Tables S1 and S2, respectively.

Network analysis

This strategy has been previously developed and applied by our group (Sacco et al, 2012, 2016). Kinase–substrate relationships and physical interactions were extracted from SIGNOR and MINT, respectively (Zanzoni et al, 2002; Perfetto et al, 2015), and were mapped onto the complete human proteome in Cytoscape (Shannon et al, 2003). Then the network was first filtered to maintain only relationships between proteins that were significantly modulated in mdx LFD FAPs compared with wt LFD FAPs and in mdx HFD FAPs compared with mdx LFD FAPs.

Flow cytometry

Immunophenotyping of Sca1+ FAPs a was performed by staining 4.0 × 10⁵ cells with anti-CD140a (APC) (PDGFRα) antibody for 30 min at 4°C. Immunophenotyping of ITGA7+ MuSCs was performed by staining 4.0 × 10⁵ cells with anti-ITGA7 (APC) antibody for 30 min at 4°C. Subsequently, MuSCs were permeabilized with 0.3% Triton-X 100 in 1× PBS for 15 min at 4°C. Permeabilized cells were incubated with anti-Pax7 (FITC) for 30 min at 4°C. The cells were washed twice and analyzed using CytoFlex Cytometer (Beckman Coulter). Data were analyzed using the Cytexpert Software (Beckman Coulter).

Freshly purified wt and mdx FAPs were resuspended in FAPs-GM and incubated in the absence/presence of 10 μM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) for 30 min at 37°C. After mitochondrial uncoupling, FAPs were incubated with 200 nM of MitoTracker Red CMXRos or MitoTracker Green FM for 20 min. FAPs were washed twice with 1× PBS and fixed in 0.5% PFA before flow cytometry analysis. For each sample, 10,000³ events were recorded using BD FACSCALIBUR (BD Biosciences).

ATP measurement

Freshly isolated FAPs were seeded at a cell density of 6 × 10⁴ cells/cm² in 96-well plates. After 20 h, ATP content was evaluated with CellTiter-Glo Luminescent Viability Assay (Promega) according to the manufacturer’s instructions. Briefly, CellTiter-Glo reagent was added at a ratio of 1:1 to cell culture medium and mixed for 2 min on an orbital shaker. Samples were transferred to a black-walled 96-well plate and luminescence was recorded through Victor Multilabel plate reader (PerkinElmer). Background consisted of FAP-GM incubated in the absence of FAPs and processed as described above. Data are presented as mean relative luminescence units.

Bioenergetics analysis of FAPs and MuSCs

Freshly isolated wt and mdx FAPs/MuSCs were plated on Seahorse XFe96 Microplates (Agilent Technologies) at the density of 1.4 × 10⁵ cells/cm² and cellular bioenergetics assessed 4 d later. Alternatively, subcultured FAPs were plated at the density of 7.0 × 10⁴. After 24 h, the cells were incubated in Cyto-Grow:RPMI in 1:1 ratio for 20 h to stimulate mitochondrial functionality before assessing the cellular bioenergetics. Cartridges were hydrated overnight with Seahorse XF Calibrant and incubated at 37°C in the absence of CO₂. Calibrant was changed and refreshed 1 h before the assay. Mitochondrial stress test was performed according to Agilent’s recommendations. Briefly, the cells were washed four times with Seahorse XF Base Medium supplemented with 10 mM glucose, 1 mM sodium pyruvate, and 2 mM glutamine (pH 7.4 ± 0.01). The cells were incubated at 37°C in the absence of CO₂ for 1 h. Mitochondrial inhibitors were sequentially injected at the following final concentrations: 1 μM oligomycin, 1.5 μM FCCP, and 1 μM/1 μM Rotenone/Antimycin (Sigma-Aldrich).

Immunoblot analysis

FAPs were washed in PBS 1× and stored at −80°C or directly lysed in ice-cold radio-immunoprecipitation assay lysis buffer (150 mM NaCl, 50 mM Tris–HCl, pH 7.5, 1% Nonidet P-40, 1 mM EGTA, 5 mM MgCl₂, and 0.1% SDS) supplemented with 1 mM PMSF, 1 mM ortovanadate, 1 mM NaF, protease inhibitor mixture 1×, inhibitor phosphatase mixture II 1×, and inhibitor phosphatase mixture III 1×. Protein lysates were separated at 15,500g for 30 min. The total protein concentration was determined using the Bradford reagent. Protein extracts were denatured and heated at 95°C for 10 min in NuPAGE LDS Sample Buffer that contained DTT as a reducing agent (NuPAGE Sample Reducing Agent). According to the needs, proteins were resolved using either 4–15% or 4–20% Bio-Rad Mini-PROTEAN TGX/CRITERION polyacrylamide gels. Proteins were transferred to Trans-Blot Turbo Mini or Midi Nitrocellulose Membranes using a Trans-Blot Turbo Transfer System (Bio-Rad), and the nonspecific binding membranes were saturated for 1 h at RT in blocking solution (5% skimmed milk powder, 0.1% Tween 20 in 1× TBS). Saturated membranes were incubated overnight with the specific primary antibodies diluted in blocking solution. Host-specific HRP-conjugated secondary antibodies were diluted in blocking solution and used for the detection of the primary antibodies. Chemiluminescent detection was performed using Clarity Western ECL Blotting Substrates (Bio-Rad) and the Las-3000 Imaging System (Fujifilm). Band densities were quantified using Fiji and normalized to the loading control. All antibodies were diluted according to the manufacturer’s instructions.

Real-time PCR

Total RNA was extracted using TRIzol from TAs, diaphragms, and FAPs. Muscles were minced in short pieces and homogenized with a Dounce tissue grinder directly in TRIzol. Before resuspension, total RNA was precipitated overnight in the presence of 10 μg of glycogen. RNA concentration was assessed using NanoDrop Lite Spectrophotometer. Total RNA (ranging between 300 and 1,000 ng) was reverse-transcribed into cDNA with PrimeScript RT Reagent Kit. qPCR reactions were carried out with SYBR Premix Ex Taq (Tli RNaseH Plus) and performed in technical duplicates for each biological repeat. Each reaction mixture (final volume of 20 μl) contained 50 ng of cDNA. Relative expression was estimated by 2^−ΔCt as described (Livak & Schmittgen, 2001). To compare multiple runs, mRNA levels were determined by the 2^−ΔΔCt method (Livak & Schmittgen, 2001). Actb and Tuba1a were used as reference genes. The full primer list is reported in Table 2.

Histological analysis

TA and diaphragm muscles were embedded in optimal cutting temperature medium and snap-frozen in liquid nitrogen. Embedded muscles were stored at −80°C before cryosectioning.

Prior optimal cutting temperature embedding, for GFP immunolabelling in transplanted TAs, muscles were fixed in 4% PFA for 4 h at RT and incubated for 4 h in sucrose gradient solutions (10%, 20% and 30%). Muscles were transverse cryosectioned at 10 μm using a Leica cryostat. Muscle sections were sequentially collected in different glass slides, thus adjacent sections were separated at least 100 μm from each other. Cryosections were performed for the entire length of the muscles.

For the hematoxylin and eosin stain (H&E), sections were fixed in 4% PFA for 10 min at RT. Fixed sections were washed twice in 1× PBS and incubated in hematoxylin solution for 20 min. Sections were rinsed for 10 min in tap water and washed in ultrapure water. Samples were incubated with the eosin solution for 1 h. Finally, stained sections were ethanol-dehydrated, clarified with the Histo-Clear solution and mounted with the Eukitt mounting medium.

Immunofluorescence

Cells and sections were fixed in 2% PFA for 15 min at RT. For immunocytochemistry fixed cells were washed twice in 1× PBS and incubated in permeabilization solution (0.5% Triton X-100 in 1× PBS) for 5 min. Unspecific binding were saturated by incubating samples in blocking solution (10% FBS and 0.1% Triton X-100 in 1× PBS) for 1 h. The cells were incubated overnight at 4°C in gentle shaking in the presence of the primary antibodies. The cells were washed four times with 0.1% Triton X-100 in 1× PBS and incubated, for 30 min at RT, with host-specific secondary antibodies. Primary and secondary antibodies were diluted according to the manufacturer’s recommendations in blocking solution. Finally, the cells were washed four times with 0.1% Triton X-100 in 1× PBS and counterstained for 5 min at RT with the Hoechst solution (1 mg/ml Hoechst 33342 and 0.1% Triton X-100 in 1× PBS). Cells were washed twice with 1× PBS and stored at 4°C.

For muscle section immunofluorescence, different protocols were adapted according to the antigen of interest. For laminin staining, fixed sections were washed twice in 1× PBS and permeabilized (0.3% Triton X-100 in 1× PBS) for 30 min at RT. Permeabilized slices were incubated in blocking solution (10% goat serum, 1% glycine, and 0.1% Triton X-100 in 1× PBS) for 1 h at RT. Blocked specimen were incubated overnight at 4°C in the presence of anti-laminin (Sigma-Aldrich) diluted 1:200 in blocking solution.

For the in situ detection of PDGFRα, fixed and permeabilized sections were blocked for 30 min in protein block serum-free reagent (DAKO) and then incubated overnight with anti-PDGFRα diluted 1:80 in a carrier solution (4% BSA in 1× PBS). After incubation with the primary antibodies, the sections were washed four times with 0.1% Triton X-100 in 1× PBS and incubated for 30 min at RT with host-specific secondary antibodies. Nuclei were revealed by incubating samples for 10 min with Hoechst solution. The sections were washed twice in 1× PBS and mounted with Aqua Poly/Mount mounting medium.

TUNEL assay

In situ detection of apoptotic nuclei was performed through the In Situ Cell Death Detection Kit, TMR red (Sigma-Aldrich). Cells were fixed with 2% PFA for 20 min at RT and permeabilized with 0.5% Triton X-100 in 1× PBS for 5 min at RT. Positive controls were treated with DNAse I (3 U/ml in 50 mM Tris–HCl, pH 7.5, and 1 mg/ml BSA) for 10 min at RT. Negative controls were treated only with the enzyme buffer without transferase enzyme. All wells were incubated with 50 μl of reaction solution for 1 h at 37°C in humidified chamber. After labelling, the samples were washed with 1× PBS and counterstained with Hoechst 33342. Images were acquired as described in “Image acquisition.”

EdU incorporation assay

For EdU labelling (Invitrogen), the cells were seeded at a concentration of 6.0 × 10³ cells/cm² and incubated in the presence of 10 μM of 5-ethylnyl-2′-deoxyuridine (EdU) the day prior fixation. Click-iT reaction was performed according to the manufacturer’s instructions.

Oil Red O (ORO)

Oil Red O (Sigma-Aldrich) stock solution was prepared according to the manufacturer’s recommendations. Fixed and permeabilized cells were washed twice with 1× PBS and incubated for 10 min with filtered ORO working solution (3:2 ratio ORO:ultrapure water). Stained cells were washed twice for 10 min with 1× PBS and counterstained using Hoechst 33342. ORO-stained cells were acquired via fluorescence microscopy.

Image acquisition

Immunolabeled cells and section were acquired using the DMI6000B fluorescent microscope (Leica). Cell Micrographs were captured in unbiased fashion with the “matrix screener” mode, by acquiring nine non-overlapping field across the well.

Section micrographs were captured with DMI6000B and Nikon Eclipse TE300 microscopes (Nikon). At least three independent fields were captured in five nonadjacent sections for each mouse. Acquisition were performed at 40×, 20×, or 10× magnification.

Representative micrographs were captured using the confocal microscope Olympus IX-81 at 20× magnitude.

For bright-field microscopy, at least three non-overlapping micrographs of five independent H&E–stained sections were captured using a Zeiss Lab A1 AX10 microscope at the 20× magnification.

Biochemical analysis

Before sacrifice, a blood sample was collected from each mouse in SST microtainers by retro-orbital sampling. For biochemical clinical analysis, blood samples were incubated for 20 min at RT and sera separated at 15,500g for 7 min. Non-fasting glucose, triglycerides, cholesterol, creatine kinase, and creatinine were measured using the automatic analyzer Keylab (BPC BioSed Srl).

Histological analysis

Centrally nucleated myofibers were counted and scored manually using Fiji (Schindelin et al, 2012) by three independent collaborators in blind analysis. Results are expressed as ratio over the total myofibers.

CSA measurements of myofibers were conducted on laminin-stained sections. The myofiber diameters were evaluated using a Fiji plug-in and data represented as average CSA and frequency distribution, for each mouse. For the pseudo-color representation of the CSA, images were processed with the ROI Color Coder macro using Fiji (http://imagejdocu.tudor.lu/doku.php?id=macro:rol_color_coder) (Kopinke et al, 2017). The manual analysis of the images was performed in blind.

Seahorse data processing

OCR was normalized by estimating the number of cells for each experimental sample. Briefly, immediately after the assay, the cells were fixed with 2% PFA for 15 min. Nuclei were labelled with Hoechst 33342 and a representative field of each well was captured and automatically scored with CellProfiler (Carpenter et al, 2006). An estimation of the total nuclei number for each condition was calculated, correlating the field to the well areas. OCR values are expressed as pmol O₂/min/10³ cells. Mitochondria activity parameters were extracted with Agilent Wave Software (version 2.6.0).

Cell differentiation measurements

Adipogenic differentiation of FAPs was estimated in unbiased fashion using CellProfiler through a dedicated pipeline that recognize, for each captured field, the ORO-positive area (expressed in pixels). The modules of the pipeline were routinely adjusted in agreement with the image magnifications. The average ORO-positive area was normalized over the average number of cells per field. Alternatively, the percentage of adipogenic differentiation was evaluated by counting the number of adipocytes over the total cells in each field. Positive objects were manually scored using Fiji by two independent collaborators.

Adipocyte dimension was evaluated by measuring the area of each lipid droplet associated with each adipocyte. Briefly, three randomly and non-overlapping field of perilipin-stained adipocytes were acquired at 40× magnification. The area of each lipid droplet was delimited using the “Frehand selection” tool of Fiji. At least 15 adipocytes for each field were included in the analysis.

Myogenic differentiation of MuSCs was scored manually using Fiji, by calculating the fusion index. Fusion index was defined as the ratio between the number of nuclei included into MyHC-expressing cells (containing at least three nuclei) over the number of nuclei for each field.

The percentage of EdU-positive cells was estimated in unbiased fashion through a dedicated pipeline using CellProfiler.

Before automated analysis, a supervised control of each CellProfiler module was performed to ensure the proper recognition of the markers of interest. The manual analysis of the images was performed in blind.

Statistical analysis

Data are represented as mean ± SEM of at least three independent experimental samples unless otherwise mentioned. In addition, cell studies were further performed at least in technical duplicates. Statistical significance between two groups was estimated using the unpaired t test assuming a two-tailed distribution. Multiple comparisons between three or more groups were performed using one-way or two-way ANOVA. Statistical significance is defined as *P < 0.05; **P < 0.01; ***P < 0.001. All statistical analyses were performed using Prism 7 (GraphPad).

Contact for reagent and resource sharing

Further information for reagents may be directed to, and will be fulfilled by the lead contact, Francesca Sacco (francesca.sacco{at}uniroma2.it).

Data and software availability

The datasets generated and/or analyzed in the current study are available from the corresponding authors on reasonable request.