Sc 8828

ASC were harvested and seeded in 24-well plates (100,000 cells/well) for 24 h in RPMI supplemented with 10% FCS. Then, 24 h later, MNC were added in the presence or absence of phytohemagglutinin (PHA, 5 µg/mL, Sigma-Aldrich). ASC/MNC ratios of 1:5 were used. For mRNA analyses, the culture period was 24 h, whereas it was 48 h when conditioning media were collected. In that latter case, IL-17A neutralizing antibodies (Secukinumab, Novartis PHArma S.A.S., 92506, Rueil-Malmaison, France) at 50 μg/mL or IFNγ-neutralizing antibodies (Invitrogen, 16-7318-81) at 50 μg/mL were eventually added from the beginning of cultures. For blocking experiments, ASC/MNC cocultures were treated with PD-L1 neutralizing antibodies (R&D Systems, Minneapolis, MN, USA; AF156) at 1 or 10 μg/mL, or with irrelevant polyclonal goat IgG (sc-8828; Santa-Cruz Biotechnology/INC, Heidelberg, Germany) at 10 μg/mL, or Secukinumab at 50 μg/mL.

Western blot analyses were performed in whole lysates of human muscle tissue and C2C12 myotubes and in isolated skeletal muscle mitochondria (wild-type and ZDF rats). Coomassie Brilliant Blue staining was used to determine total protein content. Analyses were performed using 10% polyacrylamide-SDS gels as previously described [29 (link), 30 ]. Equal protein loading was confirmed by western blotting of porin/voltage-dependent anion channel (VDAC) for isolated mitochondria and α-actin for tissue and cells. Monoclonal antibodies against ANT1 (MSA02; Mitosciences, Eugene, OR, USA; mouse IgG; dilution 1:2,000), α-actin for tissue (sc-58670; Santa Cruz, CA, USA; mouse IgM; dilution 1:10,000), β-actin for cells (A-5316; Sigma-Aldrich, St. Louis, MO, USA; mouse IgG1; dilution 1:25,000) and a polyclonal antibody against porin/VDAC (sc-8828; Santa Cruz, CA, USA; goat IgG; dilution 1:10,000) were used. For validation, we used a protein marker (Precision Plus All Blue Prestained Protein Standards from Bio-Rad Laboratories, Veenendaal, the Netherlands; no. 1610373) on the same blots. All of these commercially available antibodies showed a single distinct band at the molecular weight indicated in the datasheets. Bands of interest were detected and quantified with Odyssey Infrared Imager (LI-COR, Leusden, the Netherlands).