human fetal pulmonary fibroblasts  (ATCC)

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

Journal: Journal of Clinical Biochemistry and Nutrition

Article Title: Pulmonary activation of vitamin D 3 and preventive effect against interstitial pneumonia

doi: 10.3164/jcbn.19-48

Figure Lengend Snippet: Expression of the vitamin D receptor and vitamin D-metabolizing enzymes in human fibroblasts, and VD 3 -dependent suppression of the induction of IL-1β gene expression in pulmonary fibroblasts by bleomycin. (A) To assess gene expression in MRC-5 and MRC-5 SV1 TG1 cells, RT-PCR was performed using specific primers for the vitamin D receptor (VDR) or vitamin D-metabolizing enzymes, including CYP27A1 (27A1), CYP2R1 (2R1), and CYP27B1 (27B1). (B) MRC-5 SV1 TG1 cells were treated with 25 µg/ml bleomycin for 48 h and RT-PCR was performed using specific primers for αSMA, IL-1β, and GAPDH. (C, D) Band intensities of the PCR products were converted to numerical data by image analysis software, and the data for αSMA and IL-1β were normalized by the GAPDH value.

Article Snippet: A normal human fetal pulmonary fibroblast cell line (MRC-5) and an immortalized cell line derived from MRC-5 (MRC-5 SV1 TG1) (KAC Co., Ltd., Kyoto, Japan) were maintained in α-minimal essential medium (α-MEM) containing 10% fetal calf serum (FCS) and antibiotics (Life Technologies Japan Ltd., Tokyo, Japan) at 37°C under 5% CO 2 .

Techniques: Expressing, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Software

Journal: Journal of Clinical Biochemistry and Nutrition

Article Title: Pulmonary activation of vitamin D 3 and preventive effect against interstitial pneumonia

doi: 10.3164/jcbn.19-48

Figure Lengend Snippet: Pretreatment of pulmonary fibroblasts with VD 3 suppresses bleomycin-induced expression of pro-fibrotic genes, fibrosis markers, and inflammatory markers. (A) MRC-5 SV1 TG1 cells underwent pretreatment with VD 3 or 1,25(OH) 2 D 3 overnight and then were incubated with 25 µg/ml bleomycin for 24 h, after which RT-PCR was performed using specific primers for αSMA, COLA2, SPP1, IL-1β, TGF-β1, and β-actin (ACTB). (B–D) Band intensities of the PCR products were converted to numerical data by image analysis software, and the data for SPP1, IL-1β, and TGF-β1 were normalized by the β-actin value.

Article Snippet: A normal human fetal pulmonary fibroblast cell line (MRC-5) and an immortalized cell line derived from MRC-5 (MRC-5 SV1 TG1) (KAC Co., Ltd., Kyoto, Japan) were maintained in α-minimal essential medium (α-MEM) containing 10% fetal calf serum (FCS) and antibiotics (Life Technologies Japan Ltd., Tokyo, Japan) at 37°C under 5% CO 2 .

Techniques: Expressing, Incubation, Reverse Transcription Polymerase Chain Reaction, Software