Fig 1: Schematic representation of the osteogenic differentiation timeline (days 0–21) showing the time points where medium exchanges (M) and sample collection (day numbers in pink boxes), in triplicate, were carried out. Cell pellets were used for metabolite extraction and subsequent NMR analysis of the polar phase. Cell lysates were used for the dsDNA and calcium evaluation, whereas medium samples were used for the osteogenic markers assessment (namely, OCN and OPN). M, medium exchange; †, OPN measurement was attempted, however, results were too low and variable to be discussed, possibly due to potential high retention of the protein in the extracellular matrix. Abbreviations: dsDNA, double-stranded deoxyribonucleic acid; OCN, osteocalcin; OPN, osteopontin.
Fig 2: Schematic representation of the main metabolic fluctuations observed in this work throughout the osteogenic differentiation of hAMSCs, along with a simplified depiction of the vesicle-mediated mineralization. Metabolite names in bold indicate compounds identified (additional detected metabolites may be found in Table S1), whereas those that were observed to vary carry a schematic scale as follows: unchanged metabolic levels are represented in yellow, increases are in red, and decreases are in blue. In some cases, an indication is given if the metabolite was seen to vary only between day 0 and day 21 or before and after day 7. Three-letter code is used for amino acids. Abbreviations: ALP, alkaline phosphatase; CDP, cytidine diphosphate; CoA, coenzyme A; DAG, diacylglycerol; DHAP, dihydroxyacetone phosphate; FA, fatty acid; FAD, flavin adenine dinucleotide (oxidized); FADH2, flavin adenine dinucleotide (reduced); GAP, glyceraldehyde 3-phosphate; Gla, γ-carboxyglutamate residues; HA, calcium hydroxyapatite; IMP, inosine monophosphate; NADH, nicotinamide adenine dinucleotide (reduced); PRPP, phosphoribosyl pyrophosphate; NPP1, ectonucleotide pyrophosphatase/phosphodiesterase 1; OCN, osteocalcin; OPN, ostepontin; PHOSPHO1, phosphoethanolamine/phosphocholine phosphatase; Pi, inorganic phosphate; PPi, inorganic pyrophosphate; PtdCho, phosphatidylcholine; PtdEtn, phosphatidylethanolamine; PtdSer, phosphatidylserine. Other metabolites are abbreviated as shown in Figure 4. Some elements of this picture were adapted from Servier Medical Art (smart.servier.com) and are licensed under a Creative Commons Attribution 3.0 Unported (CC BY 3.0) license.
Fig 3: Correlations between serum Spp1 concentrations and clinical parameters of ALS. (A) Correlation between serum Spp1 and ALSFRS‐R score at sampling (r = −0.25, p = 0.02). (B) Between serum Spp1 and disease progression rate (r = 0.37, p < 0.001). ALS, amyotrophic lateral sclerosis; ALSFRS‐R, ALS Function Rating Scale‐Revised; Spp1, secreted phosphoprotein 1.
Fig 4: Kaplan–Meier curves for survival in ALS patients. The survival rate was significantly lower in patients with high Spp1 concentrations than in those with low Spp1 concentrations (p < 0.001), with a cutoff value for Spp1 set at 24.03 ng/mL. The functional endpoint was defined as death or tracheostomy with invasive ventilation. ALS, amyotrophic lateral sclerosis; Spp1, secreted phosphoprotein 1.
Fig 5: Gene expression changes in co-culture of human primary lung fibroblasts with MDA-MB-231 cells after various time points (part 1): Fold changes (±CI) compared to day 0 for (A) ACTA2, (B) FN1, (C) TNC, (D) POSTN, (E) IGF-1, (F) SPP1 measured using qPCR. Gene expression was normalized to three reference genes. The data were obtained from three replicates from two donors. * denotes minimum 2 fold change. Cq values are listed in Supplementary Table S1.
Supplier Page from Abcam for Human Osteopontin ELISA Kit