Fig. 3
Metabolic flux analysis of hDPSCs during osteogenic differentiation. Representative oxygen consumption rates (OCR) (A) and extra cellular acidification rates (ECAR) (B) profiles of OB differentiating hDPSCs, assayed by Seahorse XFe96 Analyzer, at the indicated time-points as detailed in Methods section. Oligomycin (Oligo), FCCP, Rotenone/Antimycin A (Rot/Ant.A) and 2-deoxyglucose (2-DG) were sequentially added at the indicated arrows. The histograms in C, E, show OCR and ECAR respectively normalized to the protein content. C Basal, resting OCR; Oligo, OCR measured after the addition of the ATP synthase inhibitor oligomycin; FCCP, OCR measured after the addition of the uncoupler FCCP eliciting the maximal respiratory capacity. The shown OCRs were corrected for the residual OCR measured after the addition of the CI inhibitor rotenone. D Histogram showing OCR linked to ATP turnover (OCRATP = OCRBasal − OCROligo)) and OCR Reserve (OCRReserve = OCRFCCP − OCRBasal) as inferred from the OCR values reported in (C) as described in Methods section. E Glyc (Glycolysis), resting ECAR; Reserve, difference between ECAR measured in the presence of oligomycin and under resting conditions; Capacity, ECAR measured after the addition of oligomycin and refers to the maximal glycolytic activity with the OxPhos inhibited. The shown ECARs were corrected for the 2-DG-insensitive ECAR. F OCR/ECAR ratio under basal condition. All the bar values shown in (C–F) are means ± SEM of four independent experiments (biological replicates) carried out in 3 technical replicates under each conditions; *P < 0.05; **P < 0.005; ***P < 0.0005; #P < 0.0001 (versus undifferentiated condition for (C), (E), (F))