human pulmonary fibroblast line  (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: 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