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. 2016 Feb 27;5:e10250. doi: 10.7554/eLife.10250

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© 2016, Zhao et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 6. — To label metabolites, proteins and lipids in CAFs-secreted exosomes, CAFs were cultured in RPMI with labeled ¹³C₃ pyruvate (pyr), ¹³C₅ glutamine (gln), ¹³C₆ leucine (leu), 13C₆ lysine (lys), 13C₉-phenylalanine (phe) and U-¹³C₆ glucose. After 72h of CAFs cultures, sufficient labeling was observed in metabolites, proteins and lipids contained in exosomes. Supply of metabolites to prostate cancer cells from labeled CDEs were measured under complete or deprivation medium cultures in culture media without labeling. (A) Percentage labeling (mean enrichment) observed in metabolites inside PC3 cells cultured with labeled CDEs. (n=4). Mean enrichment is calculated as $M E = (\sum_{i = 1}^{N} i \times M_{i}) / N$ . where $N$ is number of carbons in the metabolite and $M_{i}$ is abundance of (M+i) isotopologue (B) Mass fraction of heaviest labeled isotopologues of TCA cycle metabolites enriched by labeled CDEs, in prostate cancer cells cultured under complete or nutrient-deprived (without lys, phe, gln, pyr, leu) unlabeled medium (n=4). (C) Effect of CDEs on PC3 cell viability under deprivation (without lys, phe, gln, pyr, leu) conditions and exosome uptake inhibitors. CDEs rescue loss of PC3 cell proliferation under deprivation medium. CytoD, (1.5 μg/ml), heparin(50 μg/ml), and CQ (chloroquine, 20 μM) inhibited this rescue of viability under deprivation conditions n=10. (D) EIPA(25 μM) inhibited rescue of PC3 viability by CDEs under deprivation conditions, (n≥7). Data information: data are expressed as mean ± SEM,*p<0.05,**p<0.01, ***p<0.001.

DOI: http://dx.doi.org/10.7554/eLife.10250.012

Figure 6—figure supplement 1. — To label metabolites, proteins and lipids in CAFs-secreted exosomes, CAFs were cultured in RPMI with labeled ¹³C₃ pyruvate (pyr), ¹³C₅ glutamine (gln), ¹³C₆ leucine (leu), 13C₆ lysine (lys), 13C₉-phenylalanine (phe) and U-¹³C₆ glucose. After 72h of CAFs cultures, sufficient labeling was observed in metabolites, proteins and lipids contained in exosomes. Supply of metabolites to prostate cancer cells from labeled CDEs were measured under complete or deprivation medium cultures in culture media without labeling. (A) Percentage labeling (mean enrichment) observed in metabolites inside PC3 cells cultured with labeled CDEs. (n=4). Mean enrichment is calculated as $M E = (\sum_{i = 1}^{N} i \times M_{i}) / N$ . where $N$ is number of carbons in the metabolite and $M_{i}$ is abundance of (M+i) isotopologue (B) Mass fraction of heaviest labeled isotopologues of TCA cycle metabolites enriched by labeled CDEs, in prostate cancer cells cultured under complete or nutrient-deprived (without lys, phe, gln, pyr, leu) unlabeled medium (n=4). (C) Effect of CDEs on PC3 cell viability under deprivation (without lys, phe, gln, pyr, leu) conditions and exosome uptake inhibitors. CDEs rescue loss of PC3 cell proliferation under deprivation medium. CytoD, (1.5 μg/ml), heparin(50 μg/ml), and CQ (chloroquine, 20 μM) inhibited this rescue of viability under deprivation conditions n=10. (D) EIPA(25 μM) inhibited rescue of PC3 viability by CDEs under deprivation conditions, (n≥7). Data information: data are expressed as mean ± SEM,*p<0.05,**p<0.01, ***p<0.001.

DOI: http://dx.doi.org/10.7554/eLife.10250.012