*2.8. RNA Extraction and qRT-PCR*

RNA was isolated from the sorted apoptotic mpcs, non-phagocyted and phagocyted MPs, by using TRIzol Reagent (Invitrogen 15596026, Bleiswijk, Netherlands), according to the manufacturer's instructions. RNA was quantified using a NanoPhotoneter (Implen). For retro-transcription, 500 ng of RNA was used with the iScript Reverse Transcription Supermix for RT-quantitative qPCR (Bio-Rad 1708840). For qRT PCR, cDNA was diluted 1:10, and 5 μL of the diluted cDNA was loaded in a total volume of 20 μL (SYBR Green Supermix (Bio-Rad 172-5124) and run on the Bio-RAD CFX Connect Real-Time System. The relative quantification of gene expression was determined by the comparative CT method, and normalized to Cyclophiline A. Primers used were: Nfix for CTGGCTTACTTTGTCCACACTC; Nfix rev CCAGCTCTGTCACATTCCAGAC; Myogenin for CTGGGGACCCCTGAGCATTG; Myogenin rev ATCGCGCTCCTCCTGGTTGA; Cyclo A for GTGACTTTACACGCCATAATG; Cyclo A rev ACAAGATGCCAGGACCTGTAT.

### *2.9. Protein Extraction and Western Blot*

Protein extracts were obtained from cultured MPs lysed using RIPA buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% Triton-X, 0.1% sodium deoxycholate, 0.1% sodium dodecylsulphate (SDS), 150 mM NaCl, in deionised water), plus protease and phosphatase inhibitors for 30 min on ice. Then, samples were centrifuged at 11.000× *g* for 10 min at 4 ◦C, and the supernatants collected for protein quantification (DC Protein Assays Bio-Rad 5000111). 40 μg protein of each sample were denatured at 95 ◦C for 5 min using SDS PAGE sample-loading buffer (100 mM Tris pH 6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol, 10 mM dithiothreitol) and loaded into 8% SDS acrylamide gels. After electrophoresis, the protein was blotted into nitrocellulose membranes (Protran nitrocellulose transfer membrane; Whatman) for 2 h at 70 V at 4 ◦C. Membranes were then blocked for 1 h with 5% milk in Tris-buffered saline, plus 0.02% Tween20 (Sigma-Aldrich). Membranes were incubated with the primary antibodies O/N at 4 ◦C, using the following antibodies: rabbit anti-Nfix (1:1000,

Novus Biologicals NBP2-15039), mouse anti-vinculin (1:2500, Sigma-Aldrich V9131), rabbit anti-MYPT1 phosphorylated in Thr696 (1:500, SantaCruz Biotechnology sc-17556-R), and rabbit anti-Tot MYPT1 (1:500; SantaCruz Biotechnology, H-130). After incubation with the primary antibodies, the membranes were washed 3 times for 5 min and incubated with the secondary antibodies (1:10,000, IgG-HRP, Bio-Rad) for 45 min at RT, and then washed again 5 times for 5 min. Bands were revealed using ECL detection reagent (ThermoFisher), with images acquired using the ChemiDoc MP system (Bio-Rad). The Image Lab software was used to measure and quantify the bands of at least three independent western blot experiments. The obtained absolute quantity was compared with the reference band (Vinculin) and expressed in the graphs as normalized volume (Norm. Vol. Int.).

#### *2.10. Image Acquisition and Quantification*

Images were acquired with an inverted microscope (Leica-DMI6000B) equipped with Leica DFC365FX and DFC400 cameras and 20× and 40× magnification objectives. Necrotic myofibers were defined as pink pale patchy fibers, and phagocyted myofibers were defined as pink pale fibers invaded by basophilic single cells (MPs). For the quantification of CSA, analyses were done on damaged TA, which presented at least 75% of injured muscle. At least 8 pictures in different fields were taken and at least 500 myofibers were analyzed. For each condition of each experiment, at least 8 fields chosen randomly were counted. The number of labelled MPs or mpcs was calculated using the cell tracker in ImageJ software and expressed as a percentage of total MPs or mpcs. Fusion index was the number of nuclei within myotubes divided by the total number of nuclei.
