Journal: eLife

Article Title: FGF2-FGFR1 signaling regulates release of Leukemia-Protective exosomes from bone marrow stromal cells

doi: 10.7554/eLife.40033

Figure Lengend Snippet: HS-5 conditioned media (CM) was collected and separated by ultracentrifugation at 100,000 g into ECVs and CM without ECVs (-ECV). These fractions and RPMI media ± 10 ng/ml recombinant FGF2 were incubated with ( A ) K562 cells, or ( B ) MOLM14 cells with the concentrations of inhibitors as shown, and viability measured by MTS assay after 48 hr. Values were normalized to respective untreated condition. Ponatinib inhibits FGFRs around 100 nM and midostaurin inhibits FGFRs around 200 nM, and this activity blocks the protection of recombinant FGF2 completely (consistent with our previous work) and also blocks a portion of the protection of ECVs, which contain FGF2 as well as other proteins. K562 cells were incubated with media, ( C ) recombinant FGF2 10 ng/ml or ( D ) HS-5 ECVs and then treated with the indicated inhibitors ± 1000 nM PD173074 (PD). MOLM14 cells were incubated with media, ( E ) recombinant FGF2 10 ng/ml or ( F ) HS-5 ECVs and then treated with the indicated inhibitors ± 1000 nM PD173074 (PD). Viability was measured by MTS assay after 48 hr and normalized to respective untreated condition. In both cases, addition of PD has no effect with just media, but blocks protection by FGF2. PD also partially blocks protection by HS-5 ECVs, which contain FGF2. Wells were plated in triplicate and error bars indicate standard deviation.

Article Snippet: To test the role of FGF2 in ECV production in primary cells, equal numbers of murine stromal cells from Fgf2 +/+ and -/- mice ( Fgf2tm1Doe [ ]) were treated with PD173074 and ECVs quantified by Virocyt ( ).

Techniques: Recombinant, Incubation, MTS Assay, Activity Assay, Standard Deviation

Journal: eLife

Article Title: FGF2-FGFR1 signaling regulates release of Leukemia-Protective exosomes from bone marrow stromal cells

doi: 10.7554/eLife.40033

Figure Lengend Snippet: Equal numbers of HS-5 and HS-27 cells were plated in RPMI with exosome-depleted FBS for 24 hr. The ECVs were pelleted by ultracentrifugation at 100,000 g for 2 hr at four degrees C and resuspended in PBS. ECVs were quantified by ( A ) Nanosight, a nanovesicle tracking analysis, ( B ) Virocyt Virus Counter, a proprietary flow cytometry using fluorescent dyes that stain both nucleic acid and protein, or ( C ) transmission electron microscopy. ( D ) HS-5 and HS-27 exosomes were collected by sucrose-step gradient and analyzed by transmission electron microscopy. Vesicles were quantified by counting in three 2 × 2 μm areas per sample. All experiments were done in triplicate, error bars represent standard deviation, p values are indicated by *<0.05, **<0.005. HS-5 and HS-27 ECVs (P100) were obtained by ultracentrifugation (starting material, or SM), and the exosome fraction was further purified by a sucrose step-gradient. Sucrose layer interfaces (0–7.5%, 7.5–15%, 15–30%, 30–45%, and 45%-pellet) were collected, lysed and analyzed by immunoblot. Blots were probed with antibodies against exosomal markers CD9 and tsg101; cell compartment markers: fibronectin, lamin A/C, BCL-XL; as well as FGFR1 and FGF2. The lanes with highest enrichment for CD9 and tsg-101, indicating exosomes, are marked below.

Article Snippet: To test the role of FGF2 in ECV production in primary cells, equal numbers of murine stromal cells from Fgf2 +/+ and -/- mice ( Fgf2tm1Doe [ ]) were treated with PD173074 and ECVs quantified by Virocyt ( ).

Techniques: Virus, Flow Cytometry, Staining, Transmission Assay, Electron Microscopy, Standard Deviation, Purification, Western Blot

Journal: eLife

Article Title: FGF2-FGFR1 signaling regulates release of Leukemia-Protective exosomes from bone marrow stromal cells

doi: 10.7554/eLife.40033

Figure Lengend Snippet: HS-5 cells were exposed to a gradient of the FGFR inhibitors ( A ) PD173074 and ( B ) BGJ-398 for 48 hr prior to collecting CM. ECVs were pelleted by ultracentrifugation at 100,000 g and quantified by Virocyt Virus Counter. Error bars indicate standard deviation and p values are indicated by *<0.05. ( C ) HS-5 and HS-27 cells were incubated in media ± 1 µM PD173074 for 72 hr prior to collecting ECVs. ECVs were analyzed by immunoblot for FGF2. The exosome markers CD9 and tsg101 are also shown. ( D ) HS-5 cells were plated in media ± 1 µM PD173074 for 72 hr. P100 fractions were obtained by ultracentrifugation, and further fractionated on a sucrose step-gradient. The interfaces (0–7.5%, 7.5–15%, 15–30%, 30–45%, and 45%-pellet) were collected, lysed and processed by immunoblot with antibodies against the exosomal markers CD9 and tsg101 as well as FGFR1 and FGF2.

Article Snippet: To test the role of FGF2 in ECV production in primary cells, equal numbers of murine stromal cells from Fgf2 +/+ and -/- mice ( Fgf2tm1Doe [ ]) were treated with PD173074 and ECVs quantified by Virocyt ( ).

Techniques: Virus, Standard Deviation, Incubation, Western Blot

Journal: eLife

Article Title: FGF2-FGFR1 signaling regulates release of Leukemia-Protective exosomes from bone marrow stromal cells

doi: 10.7554/eLife.40033

Figure Lengend Snippet: ( A ) HS-5 cells were exposed to 500 nM PD173074 (PD) for 2, 4 and 6 hr before collection of CM and ECVs as previously described. ECVs were quantified by Virocyt. Results obtained in triplicate. Error bars indicates standard deviation. **p<0.005 ( B ) HS-5 cells were exposed to a gradient of BGJ-398 for 72 hr before isolation of ECVs from CM. ECVs were lysed and run on immunoblot to demonstrate reduction in exosome markers (CD9 and tsg-101).

Article Snippet: To test the role of FGF2 in ECV production in primary cells, equal numbers of murine stromal cells from Fgf2 +/+ and -/- mice ( Fgf2tm1Doe [ ]) were treated with PD173074 and ECVs quantified by Virocyt ( ).

Techniques: Standard Deviation, Isolation, Western Blot

Journal: eLife

Article Title: FGF2-FGFR1 signaling regulates release of Leukemia-Protective exosomes from bone marrow stromal cells

doi: 10.7554/eLife.40033

Figure Lengend Snippet: A doxycycline-inducible lentiviral shRNA targeting FGFR1 was used to create a stable HS-5 cell line. The cells were then treated with doxycycline to induce FGFR1 silencing and compared to a GIPZ lentiviral control. ( A ) Silencing of FGFR1 expression is shown by immunoblot of cell lysates. ECVs from doxycycline-treated cells were analyzed by ( B ) immunoblot or ( C ) Virocyt Virus Counter. *p<0.05. ( D ) Bone marrow was isolated from Fgf2 +/+ and -/- mice and cultured ex vivo to grow adherent marrow stroma. Equal numbers of cells were then plated, CM collected for 72 hr, and then ultracentrifuged to collect ECVs. The ECVs were quantified by Virocyt. *p<0.05. ( E ) Equal number of cultured marrow cells from Fgf2 +/+ and -/- mice were plated and then ECVs collected by ultracentrifugation and analyzed by immunoblot.

Article Snippet: To test the role of FGF2 in ECV production in primary cells, equal numbers of murine stromal cells from Fgf2 +/+ and -/- mice ( Fgf2tm1Doe [ ]) were treated with PD173074 and ECVs quantified by Virocyt ( ).

Techniques: shRNA, Control, Expressing, Western Blot, Virus, Isolation, Cell Culture, Ex Vivo

Journal: eLife

Article Title: FGF2-FGFR1 signaling regulates release of Leukemia-Protective exosomes from bone marrow stromal cells

doi: 10.7554/eLife.40033

Figure Lengend Snippet: FGFR1 siRNA pool was purchased from Thermo Fisher Scientific Dharmacon RNAi Technologies (Waltham, MA, USA). HS-5 cells were transfected with siRNAs using Lipofectamine 2000 reagent purchased from Thermo Fisher Scientific (Grand Island, NY, USA), according to manufacturer’s protocol. After 72 hr, cells were harvested, and cells and CM collected for analysis. siRNA effectively silences of FGFR1 in cells and leads to reduction in ECVs by ( A ) immunoblot and ( B ) Virocyt analysis.

Article Snippet: To test the role of FGF2 in ECV production in primary cells, equal numbers of murine stromal cells from Fgf2 +/+ and -/- mice ( Fgf2tm1Doe [ ]) were treated with PD173074 and ECVs quantified by Virocyt ( ).

Techniques: Transfection, Western Blot

Journal: eLife

Article Title: FGF2-FGFR1 signaling regulates release of Leukemia-Protective exosomes from bone marrow stromal cells

doi: 10.7554/eLife.40033

Figure Lengend Snippet: Fgf2 +/+ bone marrow was removed from donor mice and spinoculated with pMIG BCR-ABL retrovirus containing an IRES-GFP marker. The transfected bone marrow was then transplanted into lethally irradiated Fgf2 +/+ or -/- recipients. Mice were treated with 75 mg/kg/day nilotinib by oral gavage starting on day 11 of transplant. ( A ) Survival curves of untreated and nilotinib-treated Fgf2 +/+ and -/- mice. ( B ) GFP in peripheral blood was evaluated weekly and at time of euthanasia to quantify disease burden. The average GFP (percent of nucleated cells) is shown and did not differ significantly between groups indicating that all animals developed similar disease burden. Error bars indicate standard deviation. ( C ) Bone marrow cells from Fgf2 +/+ mice were spinoculated with pMIG BCR-ABL retrovirus containing GFP-IRES. The cells were then incubated with ECVs obtained from Fgf2 +/+ and -/- primary stroma cultured alone or with 500 nM PD173074. The next day the incubated cells were washed three times to remove cytokines and exosomes and plated in cytokine-free methylcellulose ± imatinib. After 8 days, colonies were counted and normalized to untreated condition. Graph shown on right. Error bars indicate standard error of the mean. *p<0.05 and **p<0.005. ( D ) Lineage-negative bone marrow cells were isolated from Fgf2 +/+ mice and cells were stained with DiO (green) tracer, washed, and immobilized on Poly-D-lysine coated chamber slides. ECVs from bone marrow stroma of Fgf2 +/+ or -/- mice were stained with DiI (red) tracer and added to the cells for a 24 hr incubation. Slides were stained with DAPI (blue) and imaged by confocal fluorescent microscopy. Movie of the z-stack images are included as and . ( E ) Model of bone marrow stromal FGF2 autocrine signaling and paracrine protection of leukemia cells by FGF2-containing exosomes.

Article Snippet: To test the role of FGF2 in ECV production in primary cells, equal numbers of murine stromal cells from Fgf2 +/+ and -/- mice ( Fgf2tm1Doe [ ]) were treated with PD173074 and ECVs quantified by Virocyt ( ).

Techniques: Marker, Transfection, Irradiation, Standard Deviation, Incubation, Cell Culture, Isolation, Staining, Microscopy

Journal: Journal of Cardiovascular Magnetic Resonance

Article Title: Reference ranges (“normal values”) for cardiovascular magnetic resonance (CMR) in adults and children: 2020 update

doi: 10.1186/s12968-020-00683-3

Figure Lengend Snippet: References, native myocardial T1 relaxation time and extracellular volume fraction (ECV)

Article Snippet: Fontana, 2012 [ ] , 1.5 T, Siemens, ShMOLLI, ECV , 27:23 , 47 ± 17.

Techniques:

Journal: Journal of Cardiovascular Magnetic Resonance

Article Title: Reference ranges (“normal values”) for cardiovascular magnetic resonance (CMR) in adults and children: 2020 update

doi: 10.1186/s12968-020-00683-3

Figure Lengend Snippet: Native myocardial T1 relaxation time and extracellular volume fraction (ECV)

Article Snippet: Fontana, 2012 [ ] , 1.5 T, Siemens, ShMOLLI, ECV , 27:23 , 47 ± 17.

Techniques: