human pulmonary fibroblasts  (PromoCell)

Journal: iScience

Article Title: Combined HIF-1α blockade and CHIR99021 treatment reverses pulmonary fibrosis via modulation endothelial-to-mesenchymal transition

doi: 10.1016/j.isci.2025.114028

Figure Lengend Snippet: Combination treatment of CHIR99021 and 2-ME reverses radiation-induced fibrotic changes, reducing persistent DNA damage in endothelial cells and fibroblasts (A) Schematic representation of irradiation and CHIR99021 treatment in HUVECs. HUVECs were treated with CHIR99021 (3 μM) after 3 days of irradiation (10 Gy). Immunofluorescence staining for phalloidin and VE-cadherin detection was performed 6 days post-irradiation (magnification: 400×). Scale bars = 20 μm. Bar graphs quantify phalloidin intensity. (B) Schematic representation of 2-ME and CHIR99021 treatment and irradiation in HUVECs. HUVECs were irradiated (10 Gy) and treated with 2-ME (0.5 μM) after 2 days, followed by CHIR99021 (3 μM) after 3 days. Immunofluorescence staining and quantification of γH2AX in HUVECs was performed 5 days post-irradiation (magnification: 400×); Scale bars = 10 μm; scale bar of cropped images = 5 μm. Dot graph quantifies the number of γH2AX foci in more than 100 cells. (C) Schematic representation of irradiation (10 Gy), 2-ME (0.5 μM), CHIR99021 (3 μM), and hrVEGF (30 ng/mL) treatment in HUVECs. Cell morphology and phalloidin immunofluorescence staining were performed 11 days post-irradiation (magnification: 200×). Scale bars = 300 μm. Bar graphs quantify phalloidin intensity. (D) Immunofluorescence staining and quantification of OCT4, Sox2, and Nanog in HUVECs 11 days post-irradiation (magnification: 400×). Scale bars = 5 μm. Dot graph quantifies OCT4, Sox2, and Nanog intensity in more than 100 cells. (E) HUVECs, irradiated for 48 h, were cultured on Matrigel-coated plates for 3 h in the presence of CHIR99021, 2-ME, CHIR99021+2-ME, or medium only as a control (magnification: 40×). Scale bars = 20 μm. Bar graphs quantify the number of tubes formed. (F) Schematic representation of irradiation (10 Gy) and 2-ME (0.5 μM) and CHIR99021 (3 μM) treatment in HPFs. Cell morphology and phalloidin immunofluorescence staining were performed 11 days post-irradiation (magnification: 200×). Scale bars = 300 μm. Bar graphs quantify phalloidin intensity (right panels). (G) Immunofluorescence staining and quantification of γH2AX in HPFs 11 days post-irradiation (magnification: 400×). Scale bars = 5 μm. Dot graph quantifies the number of γH2AX foci in more than 100 cells. (H) Immunofluorescence staining and quantification of OCT4, Sox2, and Nanog in HPFs 11 days post-irradiation (magnification: 400×). Scale bars = 5 μm. Dot graph quantifies OCT4, Sox2, and Nanog intensity in more than 100 cells. In all graphs, error bars indicate SEM. Statistical significance was assessed by one-way ANOVA for multiple comparisons. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. In graphs (D) and (G), error bars represent SD. The data shown are representative of repeated three independent experiments.

Article Snippet: Human umbilical vein endothelial cells (HUVECs; Promocell; #C-12203), human pulmonary fibroblasts (HPFs; Promocell; #C-12360), and diseased human lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPFs; Lonza; #CC-7231) were purchased and cultured using the media recommended by the respective manufacturers.

Techniques: Irradiation, Immunofluorescence, Staining, Cell Culture, Control

Journal: iScience

Article Title: Combined HIF-1α blockade and CHIR99021 treatment reverses pulmonary fibrosis via modulation endothelial-to-mesenchymal transition

doi: 10.1016/j.isci.2025.114028

Figure Lengend Snippet: Lineage tracing in Col1a2-Tomato mice demonstrates suppressed fibroblast activation and enhanced endothelial repair following combination treatment in RIPF (A) Schematic representation of collagen irradiation and drug administration in Col1a2-Tomato mice. Col1a2 Cre-ER mutant mice carry a tamoxifen-inducible Cre recombinase. To express Tomato in fibroblasts, mice carrying the tomato gene were crossed with Col1a2 Cre-ER mice. Tamoxifen was injected into mice to cause Tomato to be expressed in fibroblasts. Col1a2-Tomato mice were irradiated in the left lung with 90 Gy using a 4 mm diameter field. Col1a2-Tomato mice were treated with tamoxifen (2 mg/day) once daily for 4 days starting 1 h pre-irradiation and administered CHIR99021 (30 mg/kg) plus 2-ME (30 mg/kg) starting 4 days post-irradiation, with dosing continued every 2 days. Lung samples (n ≧ 5/group) were obtained 14 days post-irradiation from non-irradiated and irradiated mice. (B) Representative images of Hematoxylin & eosin staining, Masson trichrome staining, and Tomato immunofluorescence staining in lung tissues 14 days post-irradiation, treated with or without drug treatment (magnification, 200×). Scale bars = 20 μm. Scoring of fibrosis grade, quantification of collagen deposition, and Tomato + area are shown in the graph. (C) Immunohistochemistry staining of CD31 in the lung tissues of mice 14 days post-irradiation treated with or without drug treatment (magnification, 200×). Scale bars = 20 μm. Bar graphs quantify the CD31 area. (D) Immunofluorescence staining of CD31 (green), Tomato (red), and αSMA (white) in lung tissues of mice 14 days post-irradiation treated with or without drugs (magnification, 400×). Scale bars = 10 μm. Scale bar of cropped images = 5 μm. Quantification of the Tomato + CD31 + αSMA + area and the Tomato + CD31 + αSMA − area. (E) Immunofluorescence staining of Tomato (red) and αSMA (green) in the lung tissues of mice 14 days post-irradiation treated with or without drug treatment (magnification, 400×). Scale bars = 10 μm. Bar graphs quantify the αSMA and αSMA + Tomato + /αSMA + area. In the Ashcroft score graph (A) error bars represent SD. In all other graphs, error bars represent SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001 (one-way ANOVA for multiple comparisons). The data shown are representative of repeated two independent experiments.

Article Snippet: Human umbilical vein endothelial cells (HUVECs; Promocell; #C-12203), human pulmonary fibroblasts (HPFs; Promocell; #C-12360), and diseased human lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPFs; Lonza; #CC-7231) were purchased and cultured using the media recommended by the respective manufacturers.

Techniques: Activation Assay, Irradiation, Mutagenesis, Injection, Staining, Immunofluorescence, Immunohistochemistry

Journal: iScience

Article Title: Combined HIF-1α blockade and CHIR99021 treatment reverses pulmonary fibrosis via modulation endothelial-to-mesenchymal transition

doi: 10.1016/j.isci.2025.114028

Figure Lengend Snippet: HIF-1α deletion and CHIR99021 synergistically reduce radiation-induced fibrosis and promote endothelial repair (A) Schematic representation of collagen irradiation and drug administration in WT and Col1a2-HIF1α KO mice. Col1a2 Cre-ER mutant mice carry a tamoxifen-inducible Cre recombinase. For Tomato expression and HIF1α deletion in fibroblasts, mice carrying a loxP site in the HIF1a gene and mice carrying the Tomato gene were crossed with Col1a2 Cre-ER mice. Tamoxifen was injected into mice to induce the expression of Tomato and deletion of HIF1α in fibroblasts. WT and Col1a2-HIF1α KO mice were irradiated in the left lung with 90 Gy using a 4 mm diameter field. Col1a2-Tomato mice were treated with tamoxifen (2 mg/day) once daily for 4 days starting 6 days post-irradiation and administered CHIR99021 (30 mg/kg) starting 4 days post-irradiation, with dosing continued every 2 days. Lung samples (n ≧ 5/group) were obtained 21 days post-irradiation from non-irradiated and irradiated mice. (B) Representative images of Hematoxylin & eosin staining, Masson trichrome staining, and Tomato immunofluorescence staining in non-irradiated or irradiated lung tissues from WT and Col1a2-HIF1α KO mice, with or without drug treatment (magnification, 200×). Scale bars = 40 μm. Scoring of fibrosis grade, quantification of collagen deposition, and Tomato + area are shown in the graph. (C) Immunohistochemistry staining of CD31 in the lung tissues of mice 21 days post-irradiation (magnification, 200×). Scale bars = 10 μm. Bar graphs quantify the CD31 area. (D) Immunofluorescence staining of CD31 (green), Tomato (red), and αSMA (white) in the lung tissues of mice 21 days post-irradiation (magnification, 400×). Scale bars = 10 μm. Scale bar of cropped images = 5 μm. Quantification of the Tomato + CD31 + αSMA + area and the Tomato + CD31 + αSMA − area. (E) Immunofluorescence staining of Tomato (red) and αSMA (green in the lung tissues of mice 21 days post-irradiation (magnification, 400×). Scale bars = 10 μm. Bar graphs quantify the αSMA and αSMA + Tomato + /αSMA + area. In the Ashcroft score graph (A) error bars indicate SD. In all other graphs, error bars indicate SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001; ns: not significant (one-way ANOVA for multiple comparisons). The data shown are representative of repeated two independent experiments.

Article Snippet: Human umbilical vein endothelial cells (HUVECs; Promocell; #C-12203), human pulmonary fibroblasts (HPFs; Promocell; #C-12360), and diseased human lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPFs; Lonza; #CC-7231) were purchased and cultured using the media recommended by the respective manufacturers.

Techniques: Irradiation, Mutagenesis, Expressing, Injection, Staining, Immunofluorescence, Immunohistochemistry

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: iScience

Article Title: Combined HIF-1α blockade and CHIR99021 treatment reverses pulmonary fibrosis via modulation endothelial-to-mesenchymal transition

doi: 10.1016/j.isci.2025.114028

Figure Lengend Snippet: Combination treatment of CHIR99021 and 2-ME reverses radiation-induced fibrotic changes, reducing persistent DNA damage in endothelial cells and fibroblasts (A) Schematic representation of irradiation and CHIR99021 treatment in HUVECs. HUVECs were treated with CHIR99021 (3 μM) after 3 days of irradiation (10 Gy). Immunofluorescence staining for phalloidin and VE-cadherin detection was performed 6 days post-irradiation (magnification: 400×). Scale bars = 20 μm. Bar graphs quantify phalloidin intensity. (B) Schematic representation of 2-ME and CHIR99021 treatment and irradiation in HUVECs. HUVECs were irradiated (10 Gy) and treated with 2-ME (0.5 μM) after 2 days, followed by CHIR99021 (3 μM) after 3 days. Immunofluorescence staining and quantification of γH2AX in HUVECs was performed 5 days post-irradiation (magnification: 400×); Scale bars = 10 μm; scale bar of cropped images = 5 μm. Dot graph quantifies the number of γH2AX foci in more than 100 cells. (C) Schematic representation of irradiation (10 Gy), 2-ME (0.5 μM), CHIR99021 (3 μM), and hrVEGF (30 ng/mL) treatment in HUVECs. Cell morphology and phalloidin immunofluorescence staining were performed 11 days post-irradiation (magnification: 200×). Scale bars = 300 μm. Bar graphs quantify phalloidin intensity. (D) Immunofluorescence staining and quantification of OCT4, Sox2, and Nanog in HUVECs 11 days post-irradiation (magnification: 400×). Scale bars = 5 μm. Dot graph quantifies OCT4, Sox2, and Nanog intensity in more than 100 cells. (E) HUVECs, irradiated for 48 h, were cultured on Matrigel-coated plates for 3 h in the presence of CHIR99021, 2-ME, CHIR99021+2-ME, or medium only as a control (magnification: 40×). Scale bars = 20 μm. Bar graphs quantify the number of tubes formed. (F) Schematic representation of irradiation (10 Gy) and 2-ME (0.5 μM) and CHIR99021 (3 μM) treatment in HPFs. Cell morphology and phalloidin immunofluorescence staining were performed 11 days post-irradiation (magnification: 200×). Scale bars = 300 μm. Bar graphs quantify phalloidin intensity (right panels). (G) Immunofluorescence staining and quantification of γH2AX in HPFs 11 days post-irradiation (magnification: 400×). Scale bars = 5 μm. Dot graph quantifies the number of γH2AX foci in more than 100 cells. (H) Immunofluorescence staining and quantification of OCT4, Sox2, and Nanog in HPFs 11 days post-irradiation (magnification: 400×). Scale bars = 5 μm. Dot graph quantifies OCT4, Sox2, and Nanog intensity in more than 100 cells. In all graphs, error bars indicate SEM. Statistical significance was assessed by one-way ANOVA for multiple comparisons. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. In graphs (D) and (G), error bars represent SD. The data shown are representative of repeated three independent experiments.

Article Snippet: Human pulmonary fibroblasts (HPFs) , Promocell , Cat# C-12360.

Techniques: Irradiation, Immunofluorescence, Staining, Cell Culture, Control

Journal: Medicina

Article Title: Evaluation of TAM Receptor Targeting in Pathophysiology of Idiopathic Pulmonary Fibrosis

doi: 10.3390/medicina61101837

Figure Lengend Snippet: Relative mRNA expression levels of Axl, Tyro3, and Gas6 genes in IPF FBs and HPFs quantified by RT-qPCR. No Mer expression was detected in both fibroblast types. TBP gene was used as housekeeping gene. N = 4.

Article Snippet: Control human pulmonary fibroblasts (HPFs, C12360 , PromoCell, St. Louis, MO, USA) were cultured in 25 mM glucose DMEM supplemented with 10% FBS and 1% penicillin/streptomycin solution, and cells were used between passages 7 and 15.

Techniques: Expressing, Quantitative RT-PCR