Metabolic profile of QSCs

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Metabolic profile of QSCs Metabolic profile of QSCs. (A,B) SeaHorse assay results for OCRs of freshly isolated QSCs from (A) young (Young Q; n=6), and old (Old Q; n=4) Tg:Pax7-nGFP mice at 1×105 cells/well and (B) young (n=6) and D2PM (n=4) Tg:Pax7-nGFP mice at 2×105 cells/well, in real time under basal conditions and in response to mitochondrial inhibitors (O, oligomycin; F, FCCP; A, antimycin). Metabolic profile of QSCs. (A,B) SeaHorse assay results for OCRs of freshly isolated QSCs from (A) young (Young Q; n=6), and old (Old Q; n=4) Tg:Pax7-nGFP mice at 1×105 cells/well and (B) young (n=6) and D2PM (n=4) Tg:Pax7-nGFP mice at 2×105 cells/well, in real time under basal conditions and in response to mitochondrial inhibitors (O, oligomycin; F, FCCP; A, antimycin). Data are representative of at least two independent experiments. (C) Quantification of basal, minimal and maximal OCRs in old and D2PM QSCs normalised to basal respiration in young QSCs. Basal respiration is the value just before oligomycin injection, minimal respiration is the lowest value after oligomycin injection, and maximal respiration is the highest value after FCCP injection. All values were calculated after subtraction of non-mitochondrial respiration (first point after antimycin injection). (D) Relative ratio of CE [calculated as: 1−(Minimal/Basal OCR)] and SRC [calculated as: Maximal/Basal OCR] in QSCs as in C. (E) ECAR in milli pH per minute [mpH/min] under basal conditions in old, post-mortem and young QSCs freshly isolated from muscle. (F) Ratio of basal OCR to ECAR. OCR was measured at the same time as ECAR. (G) ATP production of satellite cells quantified by measuring the relative luminescence (relative luminescence unit; RLU). Luminescence of old QSCs (old Q), and QSCs D2PM and D4PM was normalised to that of young QSCs (Young Q), n=4 per group; all isolated from Tg:Pax7-nGFP mice. (H) Representative FACS profile (left) of young and old QSCs labelled with MitoTracker, and quantification of mean fluorescence intensity (right) of old QSCs (Old Q), and QSCs D2PM and D4PM normalised to that of young QSCs (Young Q ), n=6 per group. (I) Representative FACS profile (left) of young and old QSCs labelled with TMRE, and quantification of mean fluorescence intensity (right) as described for panel H (n=6 per group). Phycoerythrin (PE) and allophycocyanine (APC) fluorescent channels were used in H and I to read the fluorescent signal of MitoTracker and TMRE. Data are presented as mean (±s.e.m.). All P-values were calculated by using Mann–Whitney U or Student’s t-tests. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Francesca Pala et al. J Cell Sci 2018;131:jcs212977 © 2018. Published by The Company of Biologists Ltd