Journal: PLOS One

Article Title: Pulmonary fibroblasts activated by the addition of TNF-α and IL-4 enhance lymphangiogenic capacity and ameliorate lung fibrosis in an allogeneic rat model

doi: 10.1371/journal.pone.0342528

Figure Lengend Snippet: (A) IVIS imaging performed one day after cell administration showing pulmonary localization of transplanted fibroblasts. (B) Representative Masson trichrome-stained lung sections from each group (Day 0, saline (Ctrl), rPF, and rPF + CKs). For each group, whole lung images (top row) and corresponding magnified views (bottom row) are shown to highlight differences in fibrosis and tissue architecture. (C) Quantification of fibrotic area. Data are presented as mean ± SEM. Statistical analysis was performed using the Kruskal–Wallis test, followed by Dunn’s multiple comparisons test. *p < 0.05, **p < 0.01 vs. indicated groups.

Article Snippet: Human pulmonary fibroblasts were purchased from PromoCell (Heidelberg, Germany) and cultured with HFDM-1(+) medium (Cell Science & Technology, Osaka, Japan) supplemented with 5% (v/v) Newborn Calf Serum (NBCS) and 1% (v/v) Penicillin-Streptomycin (P/S).

Techniques: Imaging, Staining, Saline

Journal: PLOS One

Article Title: Pulmonary fibroblasts activated by the addition of TNF-α and IL-4 enhance lymphangiogenic capacity and ameliorate lung fibrosis in an allogeneic rat model

doi: 10.1371/journal.pone.0342528

Figure Lengend Snippet: (A) Effect of allogeneic rPF or rPF + CKs cell treatment on plasma levels of surfactant protein D (SP-D) levels. (B) Effect of allogeneic rPF or rPF + CKs cell treatment on plasma fibrinogen levels.Pulmonary fibrosis was induced in mice (Day 0), followed by treatment with saline (Ctrl), allogeneic pulmonary fibroblasts (rPF), or cytokine-enriched fibroblasts (rPF + CKs).Plasma samples were collected on day 14 and analyzed for fibrinogen concentration using ELISA.Sample sizes (animal numbers) were as follows: SP-D: Day 0 = 8, Ctrl = 8, rPF = 8, rPF + CKs = 8 Fibrinogen: Day 0 = 7, Ctrl = 7, rPF = 7, rPF + CKs = 7.

Article Snippet: Human pulmonary fibroblasts were purchased from PromoCell (Heidelberg, Germany) and cultured with HFDM-1(+) medium (Cell Science & Technology, Osaka, Japan) supplemented with 5% (v/v) Newborn Calf Serum (NBCS) and 1% (v/v) Penicillin-Streptomycin (P/S).

Techniques: Clinical Proteomics, Saline, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Extracellular Vesicles

Article Title: Myofibroblast‐Derived Extracellular Vesicles Drive Profibrotic Cascade Amplification in Pulmonary Fibrosis via the Nestin‐Rab7 Axis

doi: 10.1002/jev2.70223

Figure Lengend Snippet: Myofibroblast‐derived EVs drive profibrotic cascade amplification in pulmonary fibrosis . (a) Concentrations of EVs proteins in the bronchoalveolar lavage fluid (BALF) from mice isolated by sequential ultracentrifugation ( n = 6 per group). (b) Western blot analysis of CD63 and TSG101 expression in BALF from mice 21 days after bleomycin exposure. (c) Representative particle size and concentration distribution of EVs purified from BALF of mice isolated by sequential ultracentrifugation cell culture supernatants by nanoparticle tracking analysis (NTA). (d) Representative electron microscopic images of EVs purified from BALF of mice isolated by sequential ultracentrifugation cell culture supernatants. Scale bar = 200 nm. (e) Hematoxylin and eosin staining, Masson's trichrome staining and immunofluorescence images obtained using anti‐nestin (red), anti‐CD63 (green) antibody of lung sections from C57/BL6 mice 21 days after bleomycin exposure. Scale bars: 100 µm. (f) Quantification analysis of CD63 and nestin colocalization of lung sections from C57/BL6 mice 21 days after bleomycin exposure from (e) ( n = 6 per group). (g) Schematic overview of experimental design. Primary mouse lung fibroblasts were treated with TGF‐β (5 ng mL −1 ) for 24 h. After being washed by DMEM medium, cells were cultured in DMEM medium for 48 h and then the EVs were isolated by ultracentrifugation from their conditioned medium. Then we exposed primary mouse lung fibroblasts to obtained EVs or PBS with TGF‐β (5 ng mL −1 ) in different groups for 72 h and collected cells for analysis. Created in https://BioRender.com . (h) Immunofluorescence staining of α‐SMA (green) and dil (red) in primary mouse lung myofibroblasts treated with Dil‐labeled EVs. Control image shows Dil‐labeled EVs in PBS. Scale bars = 20 µm. (i) qPCR analysis of Acta2 mRNA expression in primary mouse lung fibroblasts treated with or without EVs in different groups for 72 h ( n = 3). (j) qPCR analysis of col1a1 mRNA expression in primary mouse lung fibroblasts treated with or without EVs in different groups for 72 h ( n = 3). (k) Immunofluorescence staining and (l) quantification analysis of primary mouse lung fibroblasts treated with or without EVs in different groups for 72 h using anti‐α‐SMA (green) antibody. Scale bars: 20 µm. (m) Immunofluorescence staining and (n) quantification analysis of primary mouse lung fibroblasts treated with or without EVs in different groups for 72 h using anti‐collagen I (red) antibody. Scale bars: 20 µm. Data are presented as the mean ± SD of three independent experiments; * p < 0.05, ** p < 0.01, *** p < 0.001; one‐way ANOVA and Tukey's multiple comparisons test.

Article Snippet: Human fetal pulmonary fibroblasts (MRC5 cells) and HEK293T cells were purchased from the American Type Culture Collection (ATCC) and were cultured in DMEM medium containing 10% FBS and 1% penicillin‐streptomycin in a humidified incubator of 5% CO2 at 37°C.

Techniques: Derivative Assay, Amplification, Isolation, Western Blot, Expressing, Concentration Assay, Purification, Cell Culture, Staining, Immunofluorescence, Labeling, Control

Journal: Journal of Extracellular Vesicles

Article Title: Myofibroblast‐Derived Extracellular Vesicles Drive Profibrotic Cascade Amplification in Pulmonary Fibrosis via the Nestin‐Rab7 Axis

doi: 10.1002/jev2.70223

Figure Lengend Snippet: Nestin knockdown attenuates the ability of EVs to promote TGF‐β‐induced myofibroblast differentiation . (a) Schematic overview of the experimental design. Primary mouse lung fibroblasts were treated with TGF‐β (5 ng mL −1 ) for 24 h. After being washed by DMEM medium, cells were cultured in DMEM medium for 48 h and then the EVs were isolated by ultracentrifugation from their conditioned medium. Then we exposed primary mouse lung fibroblasts to obtained EVs with or without TGF‐β (5 ng mL −1 ) in different groups for 72 h and collected cells for analysis. Created in https://BioRender.com . (b) qPCR analysis of Acta2 mRNA expression in primary mouse lung fibroblasts treated with EVs in different groups for 72 h ( n = 3). (c) qPCR analysis of Col1a1 mRNA expression in primary mouse lung fibroblasts treated with EVs in different groups for 72 h ( n = 3). (d) qPCR analysis of Fn1 mRNA expression in primary mouse lung fibroblasts treated with EVs in different groups for 72 h ( n = 3). Immunofluorescence staining of primary mouse lung fibroblasts treated with EVs in different groups for 72 h using (e) anti‐α‐SMA (green) antibody and (f) anti‐collagen I (red) antibody [Scale bars: 20 µm]. Quantification analyses of primary mouse lung fibroblasts treated with EVs in different groups for 72 h using (g) anti‐α‐SMA (green) antibody and (h) anti‐collagen I (red) antibody [Scale bars: 20 µm]. Data are presented as the mean ± SD of three independent experiments; * p < 0.05, ** p < 0.01, *** p < 0.001; one‐way ANOVA and Tukey's multiple comparisons test.

Article Snippet: Human fetal pulmonary fibroblasts (MRC5 cells) and HEK293T cells were purchased from the American Type Culture Collection (ATCC) and were cultured in DMEM medium containing 10% FBS and 1% penicillin‐streptomycin in a humidified incubator of 5% CO2 at 37°C.

Techniques: Knockdown, Cell Culture, Isolation, Expressing, Immunofluorescence, Staining

Journal: Journal of Extracellular Vesicles

Article Title: Myofibroblast‐Derived Extracellular Vesicles Drive Profibrotic Cascade Amplification in Pulmonary Fibrosis via the Nestin‐Rab7 Axis

doi: 10.1002/jev2.70223

Figure Lengend Snippet: Nestin knockdown inhibits EVs secretion in vitro . (a) Concentrations of EVs proteins in cell culture supernatants from primary mouse lung fibroblasts treated with or without TGF‐β (5 ng mL −1 ) ( n = 3). (b) Concentrations of EVs proteins in cell culture supernatants from Nestin‐knockdown cells and control cells treated with TGF‐β (5 ng mL −1 ) ( n = 3). (c) Western blot in Whole cell lysates (WCL) and EVs purified from cell culture supernatants of Nestin‐knockdown cells and control cells treated with TGF‐β (5 ng mL −1 ). We used β‐actin as internal control and calculated the ratio of the gray value of CD63 and TSG101 to that of β‐actin to obtain their relative expression level. (d) Representative particle size and concentration distribution of EVs purified from cell culture supernatants of Nestin‐knockdown cells and control cells treated with TGF‐β (5 ng mL −1 ) by NTA. (e) Representative electron microscopic images of EVs purified from cell culture supernatants of Nestin‐knockdown cells and control cells treated with TGF‐β (5 ng mL −1 ). Scale bar = 200 nm. (f) Representative electron microscopic images of Nestin‐knockdown cells and control cells treated with TGF‐β (5 ng mL −1 ). Scale bar = 500 nm. (g) The number of MVBs per cell profile from Figure . (h) The number of ILVs per MVB from Figure . (i) Immunofluorescence staining and (j) quantification analysis of Nestin‐knockdown cells and control cells treated with TGF‐β (5 ng mL −1 ) using anti‐CD63 (red) antibody. Scale bars: 20 µm. Data are presented as the mean ± SD of three independent experiments; * p < 0.05, ** p < 0.01, *** p < 0.001; One‐way ANOVA and Tukey's multiple comparisons test.

Article Snippet: Human fetal pulmonary fibroblasts (MRC5 cells) and HEK293T cells were purchased from the American Type Culture Collection (ATCC) and were cultured in DMEM medium containing 10% FBS and 1% penicillin‐streptomycin in a humidified incubator of 5% CO2 at 37°C.

Techniques: Knockdown, In Vitro, Cell Culture, Control, Western Blot, Purification, Expressing, Concentration Assay, Immunofluorescence, Staining

Journal: Journal of Extracellular Vesicles

Article Title: Myofibroblast‐Derived Extracellular Vesicles Drive Profibrotic Cascade Amplification in Pulmonary Fibrosis via the Nestin‐Rab7 Axis

doi: 10.1002/jev2.70223

Figure Lengend Snippet: Nestin knockdown inhibits EVs secretion by regulating Rab7 activity . (a) Immunofluorescence staining of primary mouse lung fibroblasts using anti‐Rab7 (red) and anti‐Nestin (green) antibody. Scale bars: 10 µm. (b) Immunoprecipitation was performed using an anti‐Nestin antibody, and immunoblotting of the protein levels of Rab7 in primary mouse lung fibroblasts. (c) Rab7 activity assays and (d) quantification analysis were performed in Nestin‐knockdown cells and control cells ( n = 3). (e) Western blot and (f, g) quantification analysis of CD63 and TSG101 expression in WCL and EVs purified by serial ultracentrifugation from cell culture supernatants of Nestin‐knockdown cells and control cells transfected with or without Rab7T22N ( n = 3). (h) Concentrations of EVs proteins purified by serial ultracentrifugation from cell culture supernatants from equal numbers of Nestin‐knockdown cells and control cells transfected with or without Rab7T22N ( n = 3). (i) Representative particle size and concentration distribution of EVs purified by serial ultracentrifugation from cell culture supernatants from equal numbers of Nestin‐knockdown cells and control cells transfected with or without Rab7T22N by NTA. (j) Immunofluorescence staining and (k) quantification analysis of Nestin‐knockdown and control cells using anti‐CD63 (green) antibody transfected with or without Rab7T22N. Scale bars: 20 µm. (l) Rab7 activity assays and (m) quantification analysis were performed in Nestin‐knockdown cells and control cells treated with or without CID‐1067700 ( n = 3). (n) Concentrations of EVs proteins purified by serial ultracentrifugation from cell culture supernatants from equal numbers of Nestin‐knockdown cells and control cells treated with or without CID‐1067700 ( n = 3). (o) Western blot and (p, q) quantification analysis of CD63 and TSG101 expression in WCL and EVs purified by serial ultracentrifugation from cell culture supernatants from equal numbers of Nestin‐knockdown cells and control cells treated with or without CID‐1067700 ( n = 3). Data are presented as the mean ± SD of three independent experiments; * p < 0.05, ** p < 0.01, *** p < 0.001; One‐way ANOVA and Tukey's multiple comparisons test.

Article Snippet: Human fetal pulmonary fibroblasts (MRC5 cells) and HEK293T cells were purchased from the American Type Culture Collection (ATCC) and were cultured in DMEM medium containing 10% FBS and 1% penicillin‐streptomycin in a humidified incubator of 5% CO2 at 37°C.

Techniques: Knockdown, Activity Assay, Immunofluorescence, Staining, Immunoprecipitation, Western Blot, Control, Expressing, Purification, Cell Culture, Transfection, Concentration Assay

Journal: Journal of Extracellular Vesicles

Article Title: Myofibroblast‐Derived Extracellular Vesicles Drive Profibrotic Cascade Amplification in Pulmonary Fibrosis via the Nestin‐Rab7 Axis

doi: 10.1002/jev2.70223

Figure Lengend Snippet: Nestin recruits TBC1D15 to inactivate Rab7 . (a) Immunofluorescence staining of primary mouse lung fibroblasts using anti‐Rab7, anti‐TBC1D15 and anti‐Nestin antibody. Scale bars: 10 µm. (b) Rab7 activity assays and (c) quantification analysis were performed in primary mouse lung fibroblasts with nestin knockdown and TBC1D15 knockdown ( n = 3). (d) Immunofluorescence staining and (e) colocalization analysis of primary mouse lung fibroblasts with nestin knockdown and TBC1D15 knockdown using anti‐CD63 (red) and anti‐Rab7 (green) antibody. Scale bars: 10 µm. (f) Representative particle size and concentration distribution of EVs purified by serial ultracentrifugation from cell culture supernatants from equal numbers of Nestin‐knockdown cells and control cells with or without TBC1D15 knockdown by NTA. (g) Western blot and (h, i) quantification analysis of CD63 and TSG101 expression in WCL and EVs purified by serial ultracentrifugation from cell culture supernatants from equal numbers of primary mouse lung fibroblasts with nestin knockdown and TBC1D15 knockdown ( n = 3). Data are presented as the mean ± SD of three independent experiments; * p < 0.05, ** p < 0.01, *** p < 0.001; One‐way ANOVA and Tukey's multiple comparisons test.

Article Snippet: Human fetal pulmonary fibroblasts (MRC5 cells) and HEK293T cells were purchased from the American Type Culture Collection (ATCC) and were cultured in DMEM medium containing 10% FBS and 1% penicillin‐streptomycin in a humidified incubator of 5% CO2 at 37°C.

Techniques: Immunofluorescence, Staining, Activity Assay, Knockdown, Concentration Assay, Purification, Cell Culture, Control, Western Blot, Expressing