Journal: Communications Biology
Article Title: Extracellular vesicles modulate integrin signaling and subcellular energetics to promote pulmonary lymphangioleiomyomatosis metastasis
doi: 10.1038/s42003-025-09004-9
Figure Lengend Snippet: a Rat DNA by qPCR. ( n = 3) (two-tailed unpaired t test). b – e RNA-Seq analyses ( n = 3). b Heat map. c Volcano plot. d Top enriched Reactome pathways. e Nodes of significant pathway relationships. f Expression of ECM genes by RT-qPCR. ( n = 3) (two-tailed unpaired t test). g Masson-Trichrome stain for collagen (blue). h Expression of S100A4 by (h1) immunohistochemistry and (h2) RT-qPCR ( n = 3) (two-tailed unpaired t test). i In vivo bioluminescent reporter imaging. (i1) Bioluminescence imaging of the lungs from mice injected i.v. with 621-L9 cells, 72 h. after i.v. injection of 621L9-plasma EVs, TSC2-plasma EVs, or controls. (i2) Quantification of relative luciferase unit at different time points, normalized to time zero (TSC-null EVs n = 6, TSC2 EVs n = 5, TSC-null EV-depleted plasma [EDP] n = 4, TSC2 EDP n = 3) (two-way ANOVA, significance for “column factor”). j ECM, epithelial, and fibroblast-related genes by RT-qPCR (TSC-null EVs n = 6, TSC2 EVs n = 5 for all except for S100A4; TSC-null EVs n = 6, TSC2 EVs n = 3 for S100A4) (two-tailed unpaired t test). k Plasma FAP ELISA (TSC-null EVs n = 4, TSC2 EVs n = 5) (two-tailed unpaired t test). l In vivo bioluminescent reporter imaging. (l1) Bioluminescence imaging of lungs from mice injected i.v. with 621-L9 cells, 48 h. after tumor TSC-null-EVs, TSC2 EVs, or PBS (PBS n = 3, TSC-null EVs n = 5, TSC2 EVs n = 4). (l2) Quantification of relative luciferase unit at different time points normalized to time zero (two-way ANOVA, significance for “interaction factor”). m Lung expression of FAP from mice exposed to PBS or indicated EV subtypes (asterisks mark FAP expression in tumor vs. metastasis TSC-null EVs) (PBS n = 2, all other groups n = 3). n In vivo bioluminescent reporter imaging. (n1) Bioluminescence imaging of lungs from mice injected i.v. with 621-L9 cells, 48 h. after metastasis TSC-null-EVs, TSC2 EVs, or controls (TSC-null and TSC2 EV-depleted medium [EDM] n = 3, TSC-null EVs n = 3, TSC2 EVs n = 4). (n2) Quantification of relative luciferase unit at different time points normalized to time zero (two-way ANOVA with Tukey’s multiple comparison test, significance for “interaction and column factor”). o Lung MMP activity using IVISSense MMP 750 FAST fluorescent probe from mice i.v. exposed to metastasis EVs, 24 h prior to probe injection (TSC-null EVs n = 5, TSC2 EVs n = 4). (o1) Representative fluorescent images at 6, 24, and 48 h post-MMP probe injection. (o2) Quantification of fluorescence photon flux in the chest region (two-way ANOVA with Tukey’s multiple comparison test). p Expression of FAP and S100A4 in LAM patient specimen by immunofluorescence. Scatter plots with bars showcase mean ± SEM. Box & whiskers plots showcase range and median. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.
Article Snippet: Mouse plasma FAP levels were determined by solid phase sandwich ELISA according to the manufacturer’s instructions using DuoSet Mouse FAP (R & D Systems, USA #DY8647-05).
Techniques: Two Tailed Test, RNA Sequencing, Expressing, Quantitative RT-PCR, Staining, Immunohistochemistry, In Vivo, Imaging, Injection, Clinical Proteomics, Luciferase, Enzyme-linked Immunosorbent Assay, Comparison, Activity Assay, Fluorescence, Immunofluorescence
![a Rat DNA by qPCR. ( n = 3) (two-tailed unpaired t test). b – e RNA-Seq analyses ( n = 3). b Heat map. c Volcano plot. d Top enriched Reactome pathways. e Nodes of significant pathway relationships. f Expression of ECM genes by RT-qPCR. ( n = 3) (two-tailed unpaired t test). g Masson-Trichrome stain for collagen (blue). h Expression of S100A4 by (h1) immunohistochemistry and (h2) RT-qPCR ( n = 3) (two-tailed unpaired t test). i In vivo bioluminescent reporter imaging. (i1) Bioluminescence imaging of the lungs from mice injected i.v. with 621-L9 cells, 72 h. after i.v. injection of 621L9-plasma EVs, TSC2-plasma EVs, or controls. (i2) Quantification of relative luciferase unit at different time points, normalized to time zero (TSC-null EVs n = 6, TSC2 EVs n = 5, TSC-null EV-depleted plasma [EDP] n = 4, TSC2 EDP n = 3) (two-way ANOVA, significance for “column factor”). j ECM, epithelial, and fibroblast-related genes by RT-qPCR (TSC-null EVs n = 6, TSC2 EVs n = 5 for all except for S100A4; TSC-null EVs n = 6, TSC2 EVs n = 3 for S100A4) (two-tailed unpaired t test). k Plasma <t>FAP</t> <t>ELISA</t> (TSC-null EVs n = 4, TSC2 EVs n = 5) (two-tailed unpaired t test). l In vivo bioluminescent reporter imaging. (l1) Bioluminescence imaging of lungs from mice injected i.v. with 621-L9 cells, 48 h. after tumor TSC-null-EVs, TSC2 EVs, or PBS (PBS n = 3, TSC-null EVs n = 5, TSC2 EVs n = 4). (l2) Quantification of relative luciferase unit at different time points normalized to time zero (two-way ANOVA, significance for “interaction factor”). m Lung expression of FAP from mice exposed to PBS or indicated EV subtypes (asterisks mark FAP expression in tumor vs. metastasis TSC-null EVs) (PBS n = 2, all other groups n = 3). n In vivo bioluminescent reporter imaging. (n1) Bioluminescence imaging of lungs from mice injected i.v. with 621-L9 cells, 48 h. after metastasis TSC-null-EVs, TSC2 EVs, or controls (TSC-null and TSC2 EV-depleted medium [EDM] n = 3, TSC-null EVs n = 3, TSC2 EVs n = 4). (n2) Quantification of relative luciferase unit at different time points normalized to time zero (two-way ANOVA with Tukey’s multiple comparison test, significance for “interaction and column factor”). o Lung MMP activity using IVISSense MMP 750 FAST fluorescent probe from mice i.v. exposed to metastasis EVs, 24 h prior to probe injection (TSC-null EVs n = 5, TSC2 EVs n = 4). (o1) Representative fluorescent images at 6, 24, and 48 h post-MMP probe injection. (o2) Quantification of fluorescence photon flux in the chest region (two-way ANOVA with Tukey’s multiple comparison test). p Expression of FAP and S100A4 in LAM patient specimen by immunofluorescence. Scatter plots with bars showcase mean ± SEM. Box & whiskers plots showcase range and median. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4698/pmc12634698/pmc12634698__42003_2025_9004_Fig6_HTML.jpg)