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R&D Systems mouse anti human il1α membrane form

Epithelial-derived <t>IL1α</t> drives tumor IL6/CXCL8 production. A, In vitro lung epithelial–osteosarcoma organotypic coculture. Red, mCherry-expressing osteosarcoma cells; green, phalloidin (actin); blue, DAPI. Scale bar, 50 µm. B and C, scRNA-seq analysis of coculture with Seurat-based clustering on Uniform Manifold Approximation and Projection (UMAP) with epithelial cells annotated by expression of KRT19 and osteosarcoma cells annotated by expression of COL1A1 . D, NicheNet heatmap and dot plot analysis demonstrating candidate epithelial-derived ligands (rows) and osteosarcoma-derived receptors (columns). The strength of evidence for ligand–receptor interaction is indicated by the shade of blue in the heatmap, whereas the level of expression and percentage of cells expressing ligand or receptor are noted in dot plots. E, Heatmap demonstrating the regulatory potential of epithelial-derived ligands for the top 50 osteosarcoma differentially expressed genes (coculture vs. monoculture). Red arrows, IL6 , CXCL3 , and CXCL8 , which are strongly regulated IL1 ligands. F, Dot plot of osteosarcoma and epithelial (HBEC3-KT) ligands. Note that IL6 upregulation in coculture occurs in a minority of cells. G, Stimulation with IL1α or IL1β significantly increases secretion of IL6 and CXCL8 in human osteosarcoma cells. H, Epithelial-induced [HBEC3-KT conditioned media (CM)] osteosarcoma IL6 and CXCL8 production requires IL1 signaling (IL1Ra, IL1 receptor antagonist anakinra). ANOVA with Dunnett multiple comparisons test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

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1) Product Images from "Metastasis-Initiating Osteosarcoma Subpopulations Establish Paracrine Interactions with Lung and Tumor Cells to Create a Metastatic Niche"

Article Title: Metastasis-Initiating Osteosarcoma Subpopulations Establish Paracrine Interactions with Lung and Tumor Cells to Create a Metastatic Niche

Journal: Cancer Research

doi: 10.1158/0008-5472.CAN-24-3360

Epithelial-derived IL1α drives tumor IL6/CXCL8 production. A, In vitro lung epithelial–osteosarcoma organotypic coculture. Red, mCherry-expressing osteosarcoma cells; green, phalloidin (actin); blue, DAPI. Scale bar, 50 µm. B and C, scRNA-seq analysis of coculture with Seurat-based clustering on Uniform Manifold Approximation and Projection (UMAP) with epithelial cells annotated by expression of KRT19 and osteosarcoma cells annotated by expression of COL1A1 . D, NicheNet heatmap and dot plot analysis demonstrating candidate epithelial-derived ligands (rows) and osteosarcoma-derived receptors (columns). The strength of evidence for ligand–receptor interaction is indicated by the shade of blue in the heatmap, whereas the level of expression and percentage of cells expressing ligand or receptor are noted in dot plots. E, Heatmap demonstrating the regulatory potential of epithelial-derived ligands for the top 50 osteosarcoma differentially expressed genes (coculture vs. monoculture). Red arrows, IL6 , CXCL3 , and CXCL8 , which are strongly regulated IL1 ligands. F, Dot plot of osteosarcoma and epithelial (HBEC3-KT) ligands. Note that IL6 upregulation in coculture occurs in a minority of cells. G, Stimulation with IL1α or IL1β significantly increases secretion of IL6 and CXCL8 in human osteosarcoma cells. H, Epithelial-induced [HBEC3-KT conditioned media (CM)] osteosarcoma IL6 and CXCL8 production requires IL1 signaling (IL1Ra, IL1 receptor antagonist anakinra). ANOVA with Dunnett multiple comparisons test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure Legend Snippet: Epithelial-derived IL1α drives tumor IL6/CXCL8 production. A, In vitro lung epithelial–osteosarcoma organotypic coculture. Red, mCherry-expressing osteosarcoma cells; green, phalloidin (actin); blue, DAPI. Scale bar, 50 µm. B and C, scRNA-seq analysis of coculture with Seurat-based clustering on Uniform Manifold Approximation and Projection (UMAP) with epithelial cells annotated by expression of KRT19 and osteosarcoma cells annotated by expression of COL1A1 . D, NicheNet heatmap and dot plot analysis demonstrating candidate epithelial-derived ligands (rows) and osteosarcoma-derived receptors (columns). The strength of evidence for ligand–receptor interaction is indicated by the shade of blue in the heatmap, whereas the level of expression and percentage of cells expressing ligand or receptor are noted in dot plots. E, Heatmap demonstrating the regulatory potential of epithelial-derived ligands for the top 50 osteosarcoma differentially expressed genes (coculture vs. monoculture). Red arrows, IL6 , CXCL3 , and CXCL8 , which are strongly regulated IL1 ligands. F, Dot plot of osteosarcoma and epithelial (HBEC3-KT) ligands. Note that IL6 upregulation in coculture occurs in a minority of cells. G, Stimulation with IL1α or IL1β significantly increases secretion of IL6 and CXCL8 in human osteosarcoma cells. H, Epithelial-induced [HBEC3-KT conditioned media (CM)] osteosarcoma IL6 and CXCL8 production requires IL1 signaling (IL1Ra, IL1 receptor antagonist anakinra). ANOVA with Dunnett multiple comparisons test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Techniques Used: Derivative Assay, In Vitro, Expressing

Persistent IL6 and CXCL8 production is limited to a small subpopulation of cells. A, Uniform Manifold Approximation and Projection (UMAP) of tumor cells subsetted from coculture demonstrating that IL6 and CXCL8 expression is limited to a single cluster of cells. B and C, IF of osteosarcoma cells stimulated with IL1α (72 hours) and stained for IL6 (green) and DAPI (blue). Scale bar, 50 µm. The percentage of IL6+ cells was quantified in n = 3 experiments, 100 cells/experiment. D, scRNA-seq analysis of OS-17 tibial xenograft tumors, OS-17 experimental lung metastasis, human primary tumors, and human lung metastasis samples demonstrating the heterogeneity of IL1R1 expression. E, Flow cytometry analysis quantifying surface IL1R1 expression in human osteosarcoma cell lines.

Figure Legend Snippet: Persistent IL6 and CXCL8 production is limited to a small subpopulation of cells. A, Uniform Manifold Approximation and Projection (UMAP) of tumor cells subsetted from coculture demonstrating that IL6 and CXCL8 expression is limited to a single cluster of cells. B and C, IF of osteosarcoma cells stimulated with IL1α (72 hours) and stained for IL6 (green) and DAPI (blue). Scale bar, 50 µm. The percentage of IL6+ cells was quantified in n = 3 experiments, 100 cells/experiment. D, scRNA-seq analysis of OS-17 tibial xenograft tumors, OS-17 experimental lung metastasis, human primary tumors, and human lung metastasis samples demonstrating the heterogeneity of IL1R1 expression. E, Flow cytometry analysis quantifying surface IL1R1 expression in human osteosarcoma cell lines.

Techniques Used: Expressing, Staining, Flow Cytometry

IL1α-induced IL6 and CXCL8 production requires NF-κB signaling in a subpopulation of cells in the G 1 cell-cycle phase. A, OS-17 cells were stimulated with IL1α and then fixed and stained for NF-κB (green) or IL1R1 (red) at the indicated time points. Note the nuclear translocation of NF-κB. Scale bar, 50 µm. B, IKK inhibitors IKK-16 and TPCA-1 abrogate IL1α-induced IL6 and CXCL8 secretion as measured by ELISA. n = 2 biological replicates done in technical triplicates. ANOVA with Dunnett multiple comparisons test. **, P < 0.01; ****, P < 0.0001. C, scRNA-seq analysis of OS-17 tibial xenograft tumors, OS-17 experimental lung metastasis, human primary tumors, and human lung metastasis samples demonstrating restriction of NF-κB activity (see Materials and Methods) in a small subpopulation of G 1 cells.

Figure Legend Snippet: IL1α-induced IL6 and CXCL8 production requires NF-κB signaling in a subpopulation of cells in the G 1 cell-cycle phase. A, OS-17 cells were stimulated with IL1α and then fixed and stained for NF-κB (green) or IL1R1 (red) at the indicated time points. Note the nuclear translocation of NF-κB. Scale bar, 50 µm. B, IKK inhibitors IKK-16 and TPCA-1 abrogate IL1α-induced IL6 and CXCL8 secretion as measured by ELISA. n = 2 biological replicates done in technical triplicates. ANOVA with Dunnett multiple comparisons test. **, P < 0.01; ****, P < 0.0001. C, scRNA-seq analysis of OS-17 tibial xenograft tumors, OS-17 experimental lung metastasis, human primary tumors, and human lung metastasis samples demonstrating restriction of NF-κB activity (see Materials and Methods) in a small subpopulation of G 1 cells.

Techniques Used: Staining, Translocation Assay, Enzyme-linked Immunosorbent Assay, Activity Assay

Sustained IL6 production is maintained by a small subset of hypoproliferative cells that anchor osteosarcoma cells to the lung niche. A, IF expression of IL6 (red) and CXCL8 (green) over time (8–72 hours) following stimulation with IL1α. White, proliferating cells (Ki-67); blue, DAPI. Scale bar, 50 µm. B and C, ELISA and IF quantitation demonstrating that IL6 and CXCL8 production remains constant over time, whereas IL6 and CXCL8+ expression by IF becomes progressively restricted to Ki-67− cells. ELISA, two biological replicates done in technical triplicate. Percentage of IL6, CXCL8, and Ki-67 quantified in n = 3 experiments, 100 cells/experiment. ANOVA with Dunnett multiple comparisons test. ***, P < 0.001; ****, P < 0.0001. D, IL6, CXCL8, and Ki-67 expression in NCH-OS-4 cells following stimulation with IL1α (72 hours). Scale bar, 50 µm. E, F420 murine cells stimulated with murine IL1α (72 hours) and stained with Ki-67 (red), IL6 (green), and DAPI (blue). Scale bar, 50 µm. F, Quantitation of IL6 and Ki-67 staining in NCH-OS-4 and F420 cells.

Figure Legend Snippet: Sustained IL6 production is maintained by a small subset of hypoproliferative cells that anchor osteosarcoma cells to the lung niche. A, IF expression of IL6 (red) and CXCL8 (green) over time (8–72 hours) following stimulation with IL1α. White, proliferating cells (Ki-67); blue, DAPI. Scale bar, 50 µm. B and C, ELISA and IF quantitation demonstrating that IL6 and CXCL8 production remains constant over time, whereas IL6 and CXCL8+ expression by IF becomes progressively restricted to Ki-67− cells. ELISA, two biological replicates done in technical triplicate. Percentage of IL6, CXCL8, and Ki-67 quantified in n = 3 experiments, 100 cells/experiment. ANOVA with Dunnett multiple comparisons test. ***, P < 0.001; ****, P < 0.0001. D, IL6, CXCL8, and Ki-67 expression in NCH-OS-4 cells following stimulation with IL1α (72 hours). Scale bar, 50 µm. E, F420 murine cells stimulated with murine IL1α (72 hours) and stained with Ki-67 (red), IL6 (green), and DAPI (blue). Scale bar, 50 µm. F, Quantitation of IL6 and Ki-67 staining in NCH-OS-4 and F420 cells.

Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Staining

IL1 signaling is required for osteosarcoma metastasis progression. A, Functional validation of IL1R1 CRISPR knockout by loss of epithelial-induced IL6 secretion measured by ELISA. n = 4 biological replicates done in triplicate. B, Representative hematoxylin and eosin–stained lungs from mice inoculated with OS-17 electroporated control (NG) or OS-17 IL1R1 CRISPR knockout cell lines ( B and C ). Scale bar, 2 mm. C, Number of metastatic lesions/slice quantified by a blinded examiner. n = 15 mice/condition. ANOVA with Dunnett multiple comparisons test. D and E, Representative images and quantification of the number of metastatic lesions and percentage of metastasis burden (relative to whole lung area) in mice inoculated via tail vein with OS-17 cells, then treated with vehicle (PBS) or anakinra 1 day after tumor injection. Welch t test. Scale bar, 2 mm. F and G, Representative images and quantification of the number of metastatic lesions and percentage of metastasis burden (relative to whole lung area) in mice inoculated via tail vein with F420 cells then treated with vehicle (PBS) or anakinra 1 day after tumor injection. Scale bar, 2 mm. Welch t test. **, P < 0.01; ****, P < 0.0001.

Figure Legend Snippet: IL1 signaling is required for osteosarcoma metastasis progression. A, Functional validation of IL1R1 CRISPR knockout by loss of epithelial-induced IL6 secretion measured by ELISA. n = 4 biological replicates done in triplicate. B, Representative hematoxylin and eosin–stained lungs from mice inoculated with OS-17 electroporated control (NG) or OS-17 IL1R1 CRISPR knockout cell lines ( B and C ). Scale bar, 2 mm. C, Number of metastatic lesions/slice quantified by a blinded examiner. n = 15 mice/condition. ANOVA with Dunnett multiple comparisons test. D and E, Representative images and quantification of the number of metastatic lesions and percentage of metastasis burden (relative to whole lung area) in mice inoculated via tail vein with OS-17 cells, then treated with vehicle (PBS) or anakinra 1 day after tumor injection. Welch t test. Scale bar, 2 mm. F and G, Representative images and quantification of the number of metastatic lesions and percentage of metastasis burden (relative to whole lung area) in mice inoculated via tail vein with F420 cells then treated with vehicle (PBS) or anakinra 1 day after tumor injection. Scale bar, 2 mm. Welch t test. **, P < 0.01; ****, P < 0.0001.

Techniques Used: Functional Assay, Biomarker Discovery, CRISPR, Knock-Out, Enzyme-linked Immunosorbent Assay, Staining, Control, Injection

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Journal: Cancer Research

Article Title: Metastasis-Initiating Osteosarcoma Subpopulations Establish Paracrine Interactions with Lung and Tumor Cells to Create a Metastatic Niche

doi: 10.1158/0008-5472.CAN-24-3360

Figure Lengend Snippet: Epithelial-derived IL1α drives tumor IL6/CXCL8 production. A, In vitro lung epithelial–osteosarcoma organotypic coculture. Red, mCherry-expressing osteosarcoma cells; green, phalloidin (actin); blue, DAPI. Scale bar, 50 µm. B and C, scRNA-seq analysis of coculture with Seurat-based clustering on Uniform Manifold Approximation and Projection (UMAP) with epithelial cells annotated by expression of KRT19 and osteosarcoma cells annotated by expression of COL1A1 . D, NicheNet heatmap and dot plot analysis demonstrating candidate epithelial-derived ligands (rows) and osteosarcoma-derived receptors (columns). The strength of evidence for ligand–receptor interaction is indicated by the shade of blue in the heatmap, whereas the level of expression and percentage of cells expressing ligand or receptor are noted in dot plots. E, Heatmap demonstrating the regulatory potential of epithelial-derived ligands for the top 50 osteosarcoma differentially expressed genes (coculture vs. monoculture). Red arrows, IL6 , CXCL3 , and CXCL8 , which are strongly regulated IL1 ligands. F, Dot plot of osteosarcoma and epithelial (HBEC3-KT) ligands. Note that IL6 upregulation in coculture occurs in a minority of cells. G, Stimulation with IL1α or IL1β significantly increases secretion of IL6 and CXCL8 in human osteosarcoma cells. H, Epithelial-induced [HBEC3-KT conditioned media (CM)] osteosarcoma IL6 and CXCL8 production requires IL1 signaling (IL1Ra, IL1 receptor antagonist anakinra). ANOVA with Dunnett multiple comparisons test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Primary antibodies used are as follows: rabbit anti-CXCL8 (Abcam, ab289967, IF), mouse anti-human vimentin (Abnova, SRL33, IF, AR = TE), rabbit anti-p21 [Cell Signaling Technology, 2947, IF, AR = CB], rabbit anti–NF-κB (Cell Signaling Technology, 8242, IF), hamster anti-podoplanin (Developmental Studies Hybridoma Bank, 8.1.1, AR = TE), rat anti–Ki-67 (Invitrogen, 14-5698-82, IF, AR = CB), mouse anti-human IL6 (R&D Systems, MAB2061, IF), mouse anti-human IL1α membrane form 488 (R&D Systems, FAB2001G, IF), goat anti-mouse IL1α (R&D Systems, AF-400-NA, IF, AR = TE).

Techniques: Derivative Assay, In Vitro, Expressing

Journal: Cancer Research

Article Title: Metastasis-Initiating Osteosarcoma Subpopulations Establish Paracrine Interactions with Lung and Tumor Cells to Create a Metastatic Niche

doi: 10.1158/0008-5472.CAN-24-3360

Figure Lengend Snippet: Persistent IL6 and CXCL8 production is limited to a small subpopulation of cells. A, Uniform Manifold Approximation and Projection (UMAP) of tumor cells subsetted from coculture demonstrating that IL6 and CXCL8 expression is limited to a single cluster of cells. B and C, IF of osteosarcoma cells stimulated with IL1α (72 hours) and stained for IL6 (green) and DAPI (blue). Scale bar, 50 µm. The percentage of IL6+ cells was quantified in n = 3 experiments, 100 cells/experiment. D, scRNA-seq analysis of OS-17 tibial xenograft tumors, OS-17 experimental lung metastasis, human primary tumors, and human lung metastasis samples demonstrating the heterogeneity of IL1R1 expression. E, Flow cytometry analysis quantifying surface IL1R1 expression in human osteosarcoma cell lines.

Techniques: Expressing, Staining, Flow Cytometry

Journal: Cancer Research

Article Title: Metastasis-Initiating Osteosarcoma Subpopulations Establish Paracrine Interactions with Lung and Tumor Cells to Create a Metastatic Niche

doi: 10.1158/0008-5472.CAN-24-3360

Figure Lengend Snippet: IL1α-induced IL6 and CXCL8 production requires NF-κB signaling in a subpopulation of cells in the G 1 cell-cycle phase. A, OS-17 cells were stimulated with IL1α and then fixed and stained for NF-κB (green) or IL1R1 (red) at the indicated time points. Note the nuclear translocation of NF-κB. Scale bar, 50 µm. B, IKK inhibitors IKK-16 and TPCA-1 abrogate IL1α-induced IL6 and CXCL8 secretion as measured by ELISA. n = 2 biological replicates done in technical triplicates. ANOVA with Dunnett multiple comparisons test. **, P < 0.01; ****, P < 0.0001. C, scRNA-seq analysis of OS-17 tibial xenograft tumors, OS-17 experimental lung metastasis, human primary tumors, and human lung metastasis samples demonstrating restriction of NF-κB activity (see Materials and Methods) in a small subpopulation of G 1 cells.

Techniques: Staining, Translocation Assay, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Cancer Research

Article Title: Metastasis-Initiating Osteosarcoma Subpopulations Establish Paracrine Interactions with Lung and Tumor Cells to Create a Metastatic Niche

doi: 10.1158/0008-5472.CAN-24-3360

Figure Lengend Snippet: Sustained IL6 production is maintained by a small subset of hypoproliferative cells that anchor osteosarcoma cells to the lung niche. A, IF expression of IL6 (red) and CXCL8 (green) over time (8–72 hours) following stimulation with IL1α. White, proliferating cells (Ki-67); blue, DAPI. Scale bar, 50 µm. B and C, ELISA and IF quantitation demonstrating that IL6 and CXCL8 production remains constant over time, whereas IL6 and CXCL8+ expression by IF becomes progressively restricted to Ki-67− cells. ELISA, two biological replicates done in technical triplicate. Percentage of IL6, CXCL8, and Ki-67 quantified in n = 3 experiments, 100 cells/experiment. ANOVA with Dunnett multiple comparisons test. ***, P < 0.001; ****, P < 0.0001. D, IL6, CXCL8, and Ki-67 expression in NCH-OS-4 cells following stimulation with IL1α (72 hours). Scale bar, 50 µm. E, F420 murine cells stimulated with murine IL1α (72 hours) and stained with Ki-67 (red), IL6 (green), and DAPI (blue). Scale bar, 50 µm. F, Quantitation of IL6 and Ki-67 staining in NCH-OS-4 and F420 cells.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Staining

Journal: Cancer Research

Article Title: Metastasis-Initiating Osteosarcoma Subpopulations Establish Paracrine Interactions with Lung and Tumor Cells to Create a Metastatic Niche

doi: 10.1158/0008-5472.CAN-24-3360

Figure Lengend Snippet: IL1 signaling is required for osteosarcoma metastasis progression. A, Functional validation of IL1R1 CRISPR knockout by loss of epithelial-induced IL6 secretion measured by ELISA. n = 4 biological replicates done in triplicate. B, Representative hematoxylin and eosin–stained lungs from mice inoculated with OS-17 electroporated control (NG) or OS-17 IL1R1 CRISPR knockout cell lines ( B and C ). Scale bar, 2 mm. C, Number of metastatic lesions/slice quantified by a blinded examiner. n = 15 mice/condition. ANOVA with Dunnett multiple comparisons test. D and E, Representative images and quantification of the number of metastatic lesions and percentage of metastasis burden (relative to whole lung area) in mice inoculated via tail vein with OS-17 cells, then treated with vehicle (PBS) or anakinra 1 day after tumor injection. Welch t test. Scale bar, 2 mm. F and G, Representative images and quantification of the number of metastatic lesions and percentage of metastasis burden (relative to whole lung area) in mice inoculated via tail vein with F420 cells then treated with vehicle (PBS) or anakinra 1 day after tumor injection. Scale bar, 2 mm. Welch t test. **, P < 0.01; ****, P < 0.0001.

Techniques: Functional Assay, Biomarker Discovery, CRISPR, Knock-Out, Enzyme-linked Immunosorbent Assay, Staining, Control, Injection

Journal: Cancer Research

Article Title: Metastasis-Initiating Osteosarcoma Subpopulations Establish Paracrine Interactions with Lung and Tumor Cells to Create a Metastatic Niche

doi: 10.1158/0008-5472.CAN-24-3360

Techniques: Derivative Assay, In Vitro, Expressing

Journal: Cancer Research

Article Title: Metastasis-Initiating Osteosarcoma Subpopulations Establish Paracrine Interactions with Lung and Tumor Cells to Create a Metastatic Niche

doi: 10.1158/0008-5472.CAN-24-3360

Techniques: Expressing, Staining, Flow Cytometry

Journal: Cancer Research

Article Title: Metastasis-Initiating Osteosarcoma Subpopulations Establish Paracrine Interactions with Lung and Tumor Cells to Create a Metastatic Niche

doi: 10.1158/0008-5472.CAN-24-3360

Techniques: Staining, Translocation Assay, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Cancer Research

Article Title: Metastasis-Initiating Osteosarcoma Subpopulations Establish Paracrine Interactions with Lung and Tumor Cells to Create a Metastatic Niche

doi: 10.1158/0008-5472.CAN-24-3360

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Staining

Journal: Cancer Research

Article Title: Metastasis-Initiating Osteosarcoma Subpopulations Establish Paracrine Interactions with Lung and Tumor Cells to Create a Metastatic Niche

doi: 10.1158/0008-5472.CAN-24-3360

Techniques: Functional Assay, Biomarker Discovery, CRISPR, Knock-Out, Enzyme-linked Immunosorbent Assay, Staining, Control, Injection

Transcriptomic Profiling of BHF7-Transfected Smooth Muscle Cells Demonstrates that BHF7 Targets a Core Cell Cycle Gene Network (A) Principle component analysis of saphenous vein smooth muscle cells transfected with lipofectamine RNAiMax alone (mock), siNTC, or BHF7 to a final dose of 25 nmol/L. (B) BHF7 targets a cell cycle-enriched gene network. Our previous research identified a “SMILR-dependent network.” The top 20 differentially-expressed genes identified from this network are enriched for cell cycle-associated genes. This same set of SMILR-dependent genes demonstrate significantly reduced expression when BHF7 is transfected into cells. (C) RNA-sequencing expression analysis of CENPF from NT, mock-transfected, IL1-PDGF-treated cells (mock), siNTC, or cells transfected with BHF7 to a final dose of 25 nmol/L. (D) qRT-PCR validation of RNA-sequencing data for CENPF expression after transfection as indicated. Each colored dot represents smooth muscle cells derived from a single patient. N = 4, ∗ P < 0.05, ∗∗ P < 0.01, repeated measures analysis of variance w/Dunnett’s test for multiple comparisons. (E) Gene ontology (GO), reactome, and KEGG pathway analysis of BHF7-repressed genes reveals a broad network of genes associated with cell cycle progression and mitosis. Abbreviations as in and .

Journal: JACC: Basic to Translational Science

Article Title: Small Interfering RNA Therapy Targeting the Long Noncoding RNA SMILR for Therapeutic Intervention in Coronary Artery Bypass Graft Failure

doi: 10.1016/j.jacbts.2025.101364

Figure Lengend Snippet: Transcriptomic Profiling of BHF7-Transfected Smooth Muscle Cells Demonstrates that BHF7 Targets a Core Cell Cycle Gene Network (A) Principle component analysis of saphenous vein smooth muscle cells transfected with lipofectamine RNAiMax alone (mock), siNTC, or BHF7 to a final dose of 25 nmol/L. (B) BHF7 targets a cell cycle-enriched gene network. Our previous research identified a “SMILR-dependent network.” The top 20 differentially-expressed genes identified from this network are enriched for cell cycle-associated genes. This same set of SMILR-dependent genes demonstrate significantly reduced expression when BHF7 is transfected into cells. (C) RNA-sequencing expression analysis of CENPF from NT, mock-transfected, IL1-PDGF-treated cells (mock), siNTC, or cells transfected with BHF7 to a final dose of 25 nmol/L. (D) qRT-PCR validation of RNA-sequencing data for CENPF expression after transfection as indicated. Each colored dot represents smooth muscle cells derived from a single patient. N = 4, ∗ P < 0.05, ∗∗ P < 0.01, repeated measures analysis of variance w/Dunnett’s test for multiple comparisons. (E) Gene ontology (GO), reactome, and KEGG pathway analysis of BHF7-repressed genes reveals a broad network of genes associated with cell cycle progression and mitosis. Abbreviations as in and .

Article Snippet: Six hours post-transfection, cells were quiesced for 48 hours in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Thermo Fisher Scientific) supplemented with 50 μg/mL penicillin (Invitrogen, Thermo Fisher Scientific), 50 μg/mL streptomycin (Invitrogen, Thermo Fisher Scientific) and 0.2% [v/v] fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific) and then stimulated for a further 48 hours with fresh 0.2% FBS DMEM media containing 10 ng/mL IL1α (R&D Systems 200-LA-010) and 20 ng/mL PDGF-ββ (R&D Systems 220-BB-010), and 10 μmol/L 5-ethynyl-2-deoxyuridine (EdU) (Invitrogen A10044, Thermo Fisher Scientific) where proliferation was assessed.

Techniques: Transfection, Expressing, RNA Sequencing, Quantitative RT-PCR, Biomarker Discovery, Derivative Assay

Effect of IL1α on CD26 expression in primary endometrial stromal cells. Cells were stimulated with increasing concentrations of IL1α (5–50 ng/mL) for 24 h ( A ) and 48 h ( B ) and CD26 protein levels analyzed by Western blot. Actin was used as loading control. IL1α significantly increased CD26 expression after 48 h at 20 ng/mL and 50 ng/mL. Representative immunoblots and the corresponding densitometric quantification from 3 independent experiments are shown. ( C ) Effect of IL1α and the CD26 inhibitor DPA on CD26 protein levels in stromal cells. Cells were treated with increasing concentrations of DPA and IL1α for 48 h. CD26 protein levels were analyzed by Western blot. IL1α significantly induced CD26, whereas DPA could not abrogate its effects. Unstimulated cells were set to 100% and used as a control. Each bar represents the mean ± SEM of three independent experiments performed in duplicates. ** p < 0.01; *** p < 0.001; Ctrl, control; DPA, diprotin A; n.s, not significant. Original images can be found in .

Journal: Biomolecules

Article Title: The Contribution of CD26-Negative Fibroblasts to Endometrial Scarring

doi: 10.3390/biom15101433

Figure Lengend Snippet: Effect of IL1α on CD26 expression in primary endometrial stromal cells. Cells were stimulated with increasing concentrations of IL1α (5–50 ng/mL) for 24 h ( A ) and 48 h ( B ) and CD26 protein levels analyzed by Western blot. Actin was used as loading control. IL1α significantly increased CD26 expression after 48 h at 20 ng/mL and 50 ng/mL. Representative immunoblots and the corresponding densitometric quantification from 3 independent experiments are shown. ( C ) Effect of IL1α and the CD26 inhibitor DPA on CD26 protein levels in stromal cells. Cells were treated with increasing concentrations of DPA and IL1α for 48 h. CD26 protein levels were analyzed by Western blot. IL1α significantly induced CD26, whereas DPA could not abrogate its effects. Unstimulated cells were set to 100% and used as a control. Each bar represents the mean ± SEM of three independent experiments performed in duplicates. ** p < 0.01; *** p < 0.001; Ctrl, control; DPA, diprotin A; n.s, not significant. Original images can be found in .

Article Snippet: The following materials were used: recombinant human IL1α (cat-no. 200-LA/CF; R&D Systems); anti-actin (cat-no. A3853, Sigma Aldrich), anti-mouse conjugated secondary antibody (cat-no. 7067) and anti-rabbit conjugated secondary antibody (cat-no. 7074) were all from Cell signaling technology (Frankfurt, Germany).

Techniques: Expressing, Western Blot, Control

Effects of IL1α and DPA on wound healing in vitro of primary endometrial stromal cells. ( A ) The confluent monolayer of stromal cells was disrupted by scratching with a pipette tip ( upper panel , 0 h) and incubated with DPA and IL1α. Cell-free areas were monitored 24 h after DPA and IL1α treatments by capturing images ( lower panel , 24 h). ( B ) The results are presented as percentages of the free gaps quantified with ImageJ. IL1α promoted wound healing of the stromal cells in vitro, which was inhibited by DPA treatment. Values are presented as means ± SEM of three independent experiments performed in duplicates. ** p < 0.01; Ctrl, control: DPA, diprotin A.

Journal: Biomolecules

Article Title: The Contribution of CD26-Negative Fibroblasts to Endometrial Scarring

doi: 10.3390/biom15101433

Figure Lengend Snippet: Effects of IL1α and DPA on wound healing in vitro of primary endometrial stromal cells. ( A ) The confluent monolayer of stromal cells was disrupted by scratching with a pipette tip ( upper panel , 0 h) and incubated with DPA and IL1α. Cell-free areas were monitored 24 h after DPA and IL1α treatments by capturing images ( lower panel , 24 h). ( B ) The results are presented as percentages of the free gaps quantified with ImageJ. IL1α promoted wound healing of the stromal cells in vitro, which was inhibited by DPA treatment. Values are presented as means ± SEM of three independent experiments performed in duplicates. ** p < 0.01; Ctrl, control: DPA, diprotin A.

Techniques: In Vitro, Transferring, Incubation, Control

Effects of IL1α and DPA on secretion of COL1A1 and TGF-β3 of primary endometrial stromal cells. Cells were incubated with DPA and IL1α for 48 h. Secretion of COL1A1 and TGF-β3 was quantified by ELISAs. ( A ) IL1α dose-dependently increased COL1A1 secretion, which was significantly abrogated by DPA. ( B ) IL1α decreased secretion of TGF-β3, which was significantly attenuated by DPA. Untreated cells were set to 100% and used as controls. Each bar represents the means ± SEM of three independent experiments performed in duplicates. * p ≤ 0.05; ** p < 0.01; Ctrl, control; DPA, diprotin A; COL1A1, pro-collagen 1A1.

Journal: Biomolecules

Article Title: The Contribution of CD26-Negative Fibroblasts to Endometrial Scarring

doi: 10.3390/biom15101433

Figure Lengend Snippet: Effects of IL1α and DPA on secretion of COL1A1 and TGF-β3 of primary endometrial stromal cells. Cells were incubated with DPA and IL1α for 48 h. Secretion of COL1A1 and TGF-β3 was quantified by ELISAs. ( A ) IL1α dose-dependently increased COL1A1 secretion, which was significantly abrogated by DPA. ( B ) IL1α decreased secretion of TGF-β3, which was significantly attenuated by DPA. Untreated cells were set to 100% and used as controls. Each bar represents the means ± SEM of three independent experiments performed in duplicates. * p ≤ 0.05; ** p < 0.01; Ctrl, control; DPA, diprotin A; COL1A1, pro-collagen 1A1.

Techniques: Incubation, Control

Effects of IL1α and DPA on migration and viability of primary endometrial stromal cells. ( A ) Stromal cells were incubated with DPA and IL1α for 24 h. IL1α enhanced endometrial stromal cell migration, which was inhibited by DPA. Untreated cells were set to 100% and used as a control (Ctrl). ( B ) Stromal cells were incubated with DPA and IL1α for 48 h. Cell viability was assessed by the trypan blue exclusion assay. DPA suppressed IL1α-induced cell viability. Untreated cells were set to 100% and used as a control. The values are presented as means ± SEM of three independent experiments performed in duplicates. Ctrl, control; DPA, diprotin A. * p ≤ 0.05; ** p < 0.01.

Journal: Biomolecules

Article Title: The Contribution of CD26-Negative Fibroblasts to Endometrial Scarring

doi: 10.3390/biom15101433

Figure Lengend Snippet: Effects of IL1α and DPA on migration and viability of primary endometrial stromal cells. ( A ) Stromal cells were incubated with DPA and IL1α for 24 h. IL1α enhanced endometrial stromal cell migration, which was inhibited by DPA. Untreated cells were set to 100% and used as a control (Ctrl). ( B ) Stromal cells were incubated with DPA and IL1α for 48 h. Cell viability was assessed by the trypan blue exclusion assay. DPA suppressed IL1α-induced cell viability. Untreated cells were set to 100% and used as a control. The values are presented as means ± SEM of three independent experiments performed in duplicates. Ctrl, control; DPA, diprotin A. * p ≤ 0.05; ** p < 0.01.

Techniques: Migration, Incubation, Control, Trypan Blue Exclusion Assay

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