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: Cell Stress & Chaperones

Article Title: The role of Atp2a2 -mediated calcium imbalance and endoplasmic reticulum stress in hydrocortisone-induced neurotoxicity

doi: 10.1016/j.cstres.2025.100112

Figure Lengend Snippet: Effects of RU486 and S2A1 on calcium homeostasis and mitochondrial membrane potential in HC-treated zebrafish larvae. (a) Fluorescence staining images of zebrafish larvae (120 hpf) in different treatment groups, focusing on the brain region: green fluorescence represents cytoplasmic Ca²⁺ labeled by Mag-Fluo-4 AM, red fluorescence represents mitochondrial Ca²⁺ labeled by Rhod-2, and the merged image shows the colocalization of the two. The merged image illustrates the colocalization of the two signals. (b) Quantitative analysis of the fluorescence intensities of Mag-Fluo 4 AM and Rhod-2 (n = 3 replicates, N = 3 biological replicates, 15 embryos per replicate. Data are presented as mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001). (c) Representative images of zebrafish larvae stained with JC-1 to assess mitochondrial membrane potential (ΔΨm). Red fluorescence is the aggregated state of JC-1 (normal membrane potential), and green fluorescence is the monomer of JC-1 (membrane potential depolarization), with merged images representing the balance of the two signals. (d) Quantification of the JC-1 red/green fluorescence ratio, reflecting changes in mitochondrial membrane potential across different groups (n = 3 replicates, N = 3 biological replicates, 15 embryos per replicate; data are presented as mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: To validate the ROS detection system, a positive control group was included: 15 normally developing 120 hpf larvae were treated with 1 mM H2O2 in E3 medium for 4 h. All larvae were then incubated in 40 μM DCFH-DA (MCE, USA) working solution at 28 °C in the dark for 1 h. After three washes in E3 medium, larvae were anesthetized with 0.03% MS-222 and imaged using a confocal microscope.

Techniques: Membrane, Fluorescence, Staining, Labeling

Journal: Cell Stress & Chaperones

Article Title: The role of Atp2a2 -mediated calcium imbalance and endoplasmic reticulum stress in hydrocortisone-induced neurotoxicity

doi: 10.1016/j.cstres.2025.100112

Figure Lengend Snippet: Effects of hydrocortisone on neuronal fluorescence signals and locomotor activity in zebrafish larvae. (a) Fluorescence imaging of transgenic zebrafish larvae (120 hpf). HuC-labeled neurons (green fluorescence) demonstrate the distribution and fluorescence intensity of neurons in the brain and spinal cord across different groups. (b) Quantification of neuronal fluorescence intensity. The y-axis represents fluorescence intensity. n = 3 replicates, N = 3 biological replicates, 15 embryos per replicate. Data are presented as mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001. (c) Representative behavioral heatmap of zebrafish larvae. The movement trajectories and activity distribution of juvenile fish, with color intensity reflecting movement frequency (red indicates high-frequency activity zones, and blue indicates low-frequency activity zones). (d) Quantification of locomotor speed in zebrafish larvae (n = 3 replicates, N = 3 biological replicates, 6 embryos per replicate). The y-axis represents swimming speed (mm/s), and data are presented as mean ± SEM. Significance levels: * P < 0.05, *** P < 0.001.

Article Snippet: To validate the ROS detection system, a positive control group was included: 15 normally developing 120 hpf larvae were treated with 1 mM H2O2 in E3 medium for 4 h. All larvae were then incubated in 40 μM DCFH-DA (MCE, USA) working solution at 28 °C in the dark for 1 h. After three washes in E3 medium, larvae were anesthetized with 0.03% MS-222 and imaged using a confocal microscope.

Techniques: Fluorescence, Activity Assay, Imaging, Transgenic Assay, Labeling

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: Antioxidants

Article Title: Iron Chelation Reduces Intracellular Hydroxyl Radicals in Normal Human Dermal Fibroblasts Independently of Aging

doi: 10.3390/antiox14121437

Figure Lengend Snippet: Iron accumulation and increased ROS due to replicative senescence of NHDFs. ( a ) Intracellular free Fe 2+ fluorescence staining images and comparison of free Fe 2+ fluorescence intensity. Stained cells were observed in multiple fields of view, and representative images are shown. The scale bar indicates 100 μm. The fluorescence intensities of young NHDFs (80 cells) and aged NHDFs (82 cells) obtained from three independent staining experiments ( n = 3) were compared using box-and-whisker plots. ( b ) Mitochondrial decline in aged NHDFs. Multiple fields of view of NHDF cells stained with Mito Tracker Deep Red were observed. Representative images are shown. The scale bar indicates 100 μm. The fluorescence intensities of young (81 cells) and aged (68 cells) NHDFs obtained from three independent experiments ( n = 3) were compared using box-and-whisker plots. ( c ) Increase in intracellular superoxide in aged NHDFs. Stained NHDFs were observed from multiple viewpoints, and representative images are shown. The scale bar indicates 100 μm. The fluorescence intensities of young (50 cells) and aged (73 cells) NHDFs obtained from three independent staining experiments ( n = 3) were compared using box-and-whisker plots. ( d ) Hydrogen peroxide release from NHDFs inhibited by NaN 3 . Hydrogen peroxide concentrations secreted into the culture medium over 24 h from young and aged NHDFs were measured with or without CAT inhibition. CAT was inhibited by NaN 3 (1.0 mM). Four independent measurements were performed ( n = 4), and the mean ± standard deviation is shown. ( e ) Intracellular HPF fluorescence staining image and comparison of HPF fluorescence intensity. Stained NHDFs were observed from multiple viewpoints, and representative images are shown. The scale bar indicates 100 μm. The fluorescence intensities of young (56 cells) and aged (56 cells) NHDFs obtained from three independent staining experiments ( n = 3) were compared using box-and-whisker plots. The boxes with quartiles shown in each section represent the median, 25th percentile, and 75th percentile. Whiskers indicate the maximum and minimum values within 1.5 times the interquartile range. Outliers are plotted separately. Dark boxes represent the young NHDFs, while light boxes represent the aged NHDFs. A Student’s t -test was performed, and a significance level of p < 0.05 was considered statistically significant. * indicates p < 0.05, while n.s indicates no significant difference. ( f ) The left panel shows a positive correlation between HPF fluorescence intensity in young (dark line and dots) and aged (pink line and dots) NHDFs and Fe(NO 3 ) 3 dose. The right panel shows the HPF fluorescence intensity in young (dark bars) and aged (pink bars) NHDFs as the mean and standard deviation for each Fe(NO 3 ) 3 dose. ANOVA and Tukey’s multiple comparison analysis were performed, with p < 0.05 indicating significant differences. * indicates p < 0.05. # indicates p < 0.05 vs. 0 μM. ♭ indicates p < 0.05 vs. 0 μM.

Article Snippet: To detect intracellular hydroxyl radicals, cells were stained with Hydroxyphenyl Fluorescein (HPF staining kit, Goryo Chemical., Sapporo, Japan) prepared at 50 μM in HBSS(+) for 30 min at 37 °C.

Techniques: Fluorescence, Staining, Comparison, Whisker Assay, Inhibition, Standard Deviation

Journal: Antioxidants

Article Title: Iron Chelation Reduces Intracellular Hydroxyl Radicals in Normal Human Dermal Fibroblasts Independently of Aging

doi: 10.3390/antiox14121437

Figure Lengend Snippet: Induction of hydroxyl radicals by hydrogen peroxide and its suppression by iron chelators. ( a ) HPF fluorescence intensity was measured independently three times in young NHDFs exposed to E-MEM supplemented with 0, 5, or 10 μM hydrogen peroxide for 24 h. Fluorescence intensity by hydrogen peroxide concentration is shown in box plots with quartiles. The number of cells measured for hydrogen peroxide concentrations of 0, 5, and 10 μM was 40 cells each. ( b ) Comparison of Ferro Orange fluorescence in young and aged NHDFs. Fluorescence intensity was measured in young and aged NHDFs from the control group, Fe(NO 3 ) 3 group, and Fe(NO 3 ) 3 + DFO group (114 cells per group) ( n = 5) and compared using a box plot. ( c ) Comparison of HPF fluorescence in young and aged NHDFs. Fluorescence intensity was measured in young and aged NHDFs from the control group, Fe(NO 3 ) 3 group, and Fe(NO 3 ) 3 + DFO group (56 cells per group) ( n = 3) and compared using a box plot. In each section, the dark boxes indicate the control group, the dotted boxes indicate the Fe(NO 3 ) 3 group, and the light boxes indicate the Fe(NO 3 ) 3 + DFO group. The boxes with quartiles shown in each section represent the median, 25th percentile, and 75th percentile. The whiskers indicate the upper and lower limits of the maximum values within 1.5 times the interquartile range. Outliers are plotted individually. One-way ANOVA and Tukey’s multiple comparison test were performed. A significance level of p < 0.05 was considered statistically significant. * indicates p < 0.05, and n.s indicates no significant difference.

Article Snippet: To detect intracellular hydroxyl radicals, cells were stained with Hydroxyphenyl Fluorescein (HPF staining kit, Goryo Chemical., Sapporo, Japan) prepared at 50 μM in HBSS(+) for 30 min at 37 °C.

Techniques: Fluorescence, Concentration Assay, Comparison, Control