Journal: bioRxiv

Article Title: Evolution of nasal and olfactory infection characteristics of SARS-CoV-2 variants

doi: 10.1101/2022.04.12.487379

Figure Lengend Snippet: a-f , Confocal image capturing a cross section of olfactory epithelium and olfactory bulb. SARS-CoV-2-infected young or old hamsters were examined at 6 dpi. Boxed areas highlight the infected lateral olfactory axons crossing the cribriform plate and projecting to the olfactory bulb.Images were captured with 4 μm Z-stack and exported by maximum intensity projections. OE, olfactory epithelium; OB, olfactory bulb. g , RNAscope analysis shows viral RNA in SARS-Cov-2 infected hamster OB glomeruli at 4dpi. h , Co-staining of Caspase-3 and Iba1 in olfactory bulb at 4dpi. h , i , Confocal image shows co-staining of endothelial cell marker CD31 and ACE2 in mouse ( h ) or hamster ( i , ACE2 only) olfactory bulb. j , Immunostaining of CD45 and microglia marker Iba1 in the olfactory bulb of hACE2 mice at 6 dpi. Arrowheads indicate Iba1 negative immune cells. In the hACE2 strain, human ACE2 overexpression was driven by mouse Krt18 promoter. Scale bars, 100 μm ( a , d ); 50 μm ( h - j ).

Article Snippet: The following primary antibodies was used: Rabbit anti-SARS-CoV-2 Nucleoprotein (1:200, Novus, NB100–56576), Rabbit anti- SARS-CoV-2 Nucleoprotein (1:500, GeneTex, GTX135357), Rabbit anti- SARS-CoV Nucleoprotein (1:1000, Rockland, 200–401-A50), Rabbit anti- SARS-CoV-2 Spike S (1:200, Sino Biological, 40150-R007), Goat anti-ACE2 (1:100, R&D, AF933, for human samples), Rabbit anti-ACE2 (1:100, Thermo, MA5–32307, for hamster samples), Goat anti-Neuropilin-1 (1:200, R&D, AF566) Mouse anti-Keratin 18 (1:500, Novus, NB500–306), Goat anti OMP (1:500, Wako, 544–10001), Mouse anti-aSMA (1:100, R&D MAB1420); Chicken anti-Vimentin (Novus NB300–223); Goat anti-Foxj1 (1:200, R&D AF3619); Mouse anti-NeuN (1:1000, BioLegend, 834502); Rat anti-CD45 (1:300, Ebioscience, 14-0451-81), Rat anti-CD31(1:50, BD, 550274), Rabbit anti-Krt5 (1:500, Covance, PRB-160P), Chicken anti-Krt5 (1:500, BioLegend, 905904), Mouse anti Tuj1 (1:300, BioLegend, 801203), Rabbit anti Iba1 (1:500, Wako, 019–19741), Rabbit anti-Cleaved Caspase-3 (1:300, Cell signaling, 9664), Rat anti Dectin2 (1:200, Bio-Rad, MCA2415T), and Goat anti-CXCL10(1:100, R&D, AF-466-NA).

Techniques: Infection, RNAscope, Staining, Marker, Immunostaining, Over Expression

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 regulate p -smad2/3, histone modifications, and pluripotent factor (A) Immunofluorescence staining of p -smad2/3 (green) and Tomato (red) in non-irradiated or irradiated lung tissues from WT and Col1a2-HIF1α KO mice 21 days post-irradiation (magnification, 400×). Scale bars = 20 μm. Scale bar of cropped images = 5 μm. Quantification of the p -smad2/3 + Tomato + /Tomato + area. (B) Immunofluorescence staining of histone modifications in lung tissues. Representative images of H3K27ac (green), H3K9ac (green), H3K9me3 (green), and Tomato (red) in the lung tissues of 21 days post-irradiation, treated with or without drug treatment (magnification, 400×) (right panel). Scale bars = 10 μm. Bar graphs quantify the H3K27ac, H3K9ac, and H3K9me3 area per nucleus area (left panel). (C) Immunofluorescence staining of OCT4 (green) and Tomato (red) 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 OCT4 + Tomato + /Tomato + area. (D) Uniform Manifold Approximation and Projection (UMAP) plot of Single-Cell Transcriptomes. The plot was generated with single-cell RNA sequencing data using scanpy package. Each sample is represented by a distinct color: ‘WT, IR’ in blue and ‘Col1a2-HIF1α KO+ CHIR99021, IR’ in orange (left panel). Comparison of endothelial- and mesenchymal-related gene expression between two conditions: ‘WT, IR’ and ‘Col1a2-HIF1α KO + CHIR99021, IR’. The bar plot represents the ratio of positive cells expressing each gene. The y-axis lists genes associated with endothelium or mesenchyme, while the x-axis represents the ratio of positive cells between the two conditions (right panel). All graph 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).

Article Snippet: IHC and IF staining were carried out using primary antibodies against CD31 (1:200; R&D Systems; #AF3628, 1:200; Abcam; #ab225883), αSMA (1:1000; Sigma-Aldrich; #A5228), γH2AX (1:200; Cell signaling; #2577), H3K27ac (1:500; Abcam; #ab4729), H3K9ac (1:500; Abcam; #ab12179), H3K9me3 (1:500; Abcam; #ab8898), p-smad2/3 (1:200; Cell signaling; #8828), OCT4 (1:200; Abcam, #ab19857), pro-SPC (1:500; Abcam, #ab90716), KRT5 (1:200; Abcam, #ab128190), HIF1α (1:200; Cayman, #10006421), Tomato (1:2000; LSbio; #LS-C340696), β-Catenin (1:100; Cell signaling; #8480), CA9 (1:200; Novus; # NB100-417) and Podocalyxin (1:200; R&D; AF1556).

Techniques: Immunofluorescence, Staining, Irradiation, Generated, RNA Sequencing, Comparison, Gene Expression, 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: IHC and IF staining were carried out using primary antibodies against CD31 (1:200; R&D Systems; #AF3628, 1:200; Abcam; #ab225883), αSMA (1:1000; Sigma-Aldrich; #A5228), γH2AX (1:200; Cell signaling; #2577), H3K27ac (1:500; Abcam; #ab4729), H3K9ac (1:500; Abcam; #ab12179), H3K9me3 (1:500; Abcam; #ab8898), p-smad2/3 (1:200; Cell signaling; #8828), OCT4 (1:200; Abcam, #ab19857), pro-SPC (1:500; Abcam, #ab90716), KRT5 (1:200; Abcam, #ab128190), HIF1α (1:200; Cayman, #10006421), Tomato (1:2000; LSbio; #LS-C340696), β-Catenin (1:100; Cell signaling; #8480), CA9 (1:200; Novus; # NB100-417) and Podocalyxin (1:200; R&D; AF1556).

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: Combination therapy of 2-ME and CHIR99021 attenuates RIPF, reducing fibrotic change of endothelial cells and modulating histone modifications (A) Schematic representation of irradiation and drug administration in mice. C57BL/6 mice were irradiated in the left lung with 90 Gy using a 4 mm diameter field. C57BL/6 mice were administered CHIR99021 (30 mg/kg) plus 2-ME (30 mg/kg) starting 4 days after irradiation, with dosing continued every 2 days. Lung samples (n ≧ 5/group) were obtained 14 days post-irradiation from non-irradiated and irradiated mice. Representative images of Hematoxylin & eosin staining and Masson trichrome staining in lung tissues 14 days post-irradiation treated with or without drug treatment (magnification, 200×). Scale bars = 20 μm. Scoring of fibrosis grade and quantification of collagen deposition are shown in the graph. (B) Immunofluorescence staining of CD31 (green) and αSMA (red) in lung tissues (magnification, 400×). Scale bars = 10 μm. Scale bar of cropped images = 5 μm. Quantification of the CD31 + αSMA + /CD31 + area and the αSMA + area. (C) Immunohistochemistry staining of γH2AX in the lung tissues of C57BL/6 mice 14 days post-irradiation treated with or without drug treatment (magnification, 400×) (right panel). Scale bars = 10 μm. Bar graphs quantify the γH2AX area per nucleus area (left panel). (D) Immunofluorescence staining of histone modifications in lung tissues. Representative images of H3K27ac (red), H3K9ac (red), and H3K9me3 (green) in the lung tissues of C57BL/6 mice 14 days post-irradiation treated with or without drug treatment (magnification, 400×) (right panel). Scale bars = 10 μm. Bar graphs quantify the H3K27ac, H3K9ac, and H3K9me3 area per nucleus (left panel). 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; ns: not significant (one-way ANOVA for multiple comparisons). The data shown are representative of two independent experiments.

Article Snippet: IHC and IF staining were carried out using primary antibodies against CD31 (1:200; R&D Systems; #AF3628, 1:200; Abcam; #ab225883), αSMA (1:1000; Sigma-Aldrich; #A5228), γH2AX (1:200; Cell signaling; #2577), H3K27ac (1:500; Abcam; #ab4729), H3K9ac (1:500; Abcam; #ab12179), H3K9me3 (1:500; Abcam; #ab8898), p-smad2/3 (1:200; Cell signaling; #8828), OCT4 (1:200; Abcam, #ab19857), pro-SPC (1:500; Abcam, #ab90716), KRT5 (1:200; Abcam, #ab128190), HIF1α (1:200; Cayman, #10006421), Tomato (1:2000; LSbio; #LS-C340696), β-Catenin (1:100; Cell signaling; #8480), CA9 (1:200; Novus; # NB100-417) and Podocalyxin (1:200; R&D; AF1556).

Techniques: Irradiation, 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: Combined treatment of 2-ME and CHIR99021 reduces fibrotic progression and DNA damage in IPF primary cells (A) Schematic representation of 2-ME (0.5 μM) and CHIR99021 (3 μM) treatment in IPF primary cells. These cells were treated with 2-ME (0.5 μM) and CHIR99021 (3 μM), 3 times every 3 days. Cell morphology and phalloidin immunofluorescence staining were assessed 7 days after drug treatment (magnification: 200×). Scale bars = 250 μm. Bar graphs quantify phalloidin intensity. (B) Immunofluorescence staining and quantification of γH2AX in IPFs 7 days after drug treatment (magnification: 400×). Scale bars = 5 μm. Dot graph quantifies the number of γH2AX foci in more than 100 cells. (C) Immunofluorescence staining and quantification of collagen1 in IPFs 7 days after treatment with DMSO or drugs (magnification: 400×). Scale bars = 10 μm. Bar graphs quantify the collagen1 intensity. (D) Representative images of cell contraction assay using matrix gel in IPFs 7 days after treatment with DMSO or drugs. The diameter of the matrix gel was measured every hour for 4 h. Scale bars = 50 mm. Graph quantifies cell contraction over 4 h. (E) RT-qPCR analysis of Vimentin and S1004a in IPFs treated with DMSO or drugs. (F) Flow-cytometric analysis of EdU incorporation in IPFs treated with DMSO or drugs. For graphs (D), (E), and (F), the error bars represent the SD of three independent experiments. In graph (B), error bars represent SD. In graphs (A) and (C), error bars indicate SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001. Graphs ((D) and (E) were analyzed by two-way ANOVA for multiple comparisons; graph (F) by one-way ANOVA for multiple comparisons; others: all others by Student’s t test. The data shown are representative of repeated three independent experiments.

Article Snippet: IHC and IF staining were carried out using primary antibodies against CD31 (1:200; R&D Systems; #AF3628, 1:200; Abcam; #ab225883), αSMA (1:1000; Sigma-Aldrich; #A5228), γH2AX (1:200; Cell signaling; #2577), H3K27ac (1:500; Abcam; #ab4729), H3K9ac (1:500; Abcam; #ab12179), H3K9me3 (1:500; Abcam; #ab8898), p-smad2/3 (1:200; Cell signaling; #8828), OCT4 (1:200; Abcam, #ab19857), pro-SPC (1:500; Abcam, #ab90716), KRT5 (1:200; Abcam, #ab128190), HIF1α (1:200; Cayman, #10006421), Tomato (1:2000; LSbio; #LS-C340696), β-Catenin (1:100; Cell signaling; #8480), CA9 (1:200; Novus; # NB100-417) and Podocalyxin (1:200; R&D; AF1556).

Techniques: Immunofluorescence, Staining, Contraction Assay, Quantitative RT-PCR