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GPR68 activation or overexpression exacerbates endothelial inflammation. ( A ) Cells were treated with either 25 ng/ml LPS, 10 µM Ogerin, or both for 6 h followed by western blot analysis to detect ICAM-1 protein levels. * p < 0.05, vs. control and ** p < 0.05, versus LPS, n = 3. ( B ) Cells were transfected with wild type (WT) or inactive mutant (Mut) GPR68 plasmids for 24 h followed by stimulation with 50 ng/ml of LPS for 1 or 6 h. Protein expression of phospho-NFkB, VCAM-1, and ICAM-1 were analyzed by western blotting. Probing for GFP was used as a <t>transfection</t> control. * p < 0.05, versus WT, n = 3 ( C ) Cells were transfected with empty vector (EV) or GPR68 wild type (WT) plasmids for 24 h and exposed to 50 ng/ml of LPS for 3 h. qPCR was performed to determine mRNA expression levels of TNF-α (upper panel), IL-6, IL-1β (middle panel), and IL-8, E-selectin (lower panel). * p < 0.05, versus control and ** p < 0.05, WT versus EV, n = 3. ( C ) Cells were transfected with GPR68 WT or inactive mutant (GPR Mut) plasmids for 24 h followed by stimulation with indicated dosed of LPS (3 h). mRNA levels of TNF-α (upper panel) or IL-6, IL-8, IL-1β, E-selectin, and CXCL5 (lower panel) were determined by real-time PCR. * p < 0.05, versus WT, n = 4. (E) Cells were subjected to non-specific (NS) or GPR68-targeting (siGPR68) siRNA transfection for 72 h and then stimulated with LPS for 6 h. Western blot analysis was carried out to measure the protein levels of phospho-NFkB, VCAM-1, and ICAM-1. Efficiency of siRNA-mediated knockdown was verified by probing the blots with GPR68; β-actin was used as a loading control. Shown are representative results of 5 independent experiments.
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GPR68 activation or overexpression exacerbates endothelial inflammation. ( A ) Cells were treated with either 25 ng/ml LPS, 10 µM Ogerin, or both for 6 h followed by western blot analysis to detect ICAM-1 protein levels. * p < 0.05, vs. control and ** p < 0.05, versus LPS, n = 3. ( B ) Cells were transfected with wild type (WT) or inactive mutant (Mut) GPR68 plasmids for 24 h followed by stimulation with 50 ng/ml of LPS for 1 or 6 h. Protein expression of phospho-NFkB, VCAM-1, and ICAM-1 were analyzed by western blotting. Probing for GFP was used as a <t>transfection</t> control. * p < 0.05, versus WT, n = 3 ( C ) Cells were transfected with empty vector (EV) or GPR68 wild type (WT) plasmids for 24 h and exposed to 50 ng/ml of LPS for 3 h. qPCR was performed to determine mRNA expression levels of TNF-α (upper panel), IL-6, IL-1β (middle panel), and IL-8, E-selectin (lower panel). * p < 0.05, versus control and ** p < 0.05, WT versus EV, n = 3. ( C ) Cells were transfected with GPR68 WT or inactive mutant (GPR Mut) plasmids for 24 h followed by stimulation with indicated dosed of LPS (3 h). mRNA levels of TNF-α (upper panel) or IL-6, IL-8, IL-1β, E-selectin, and CXCL5 (lower panel) were determined by real-time PCR. * p < 0.05, versus WT, n = 4. (E) Cells were subjected to non-specific (NS) or GPR68-targeting (siGPR68) siRNA transfection for 72 h and then stimulated with LPS for 6 h. Western blot analysis was carried out to measure the protein levels of phospho-NFkB, VCAM-1, and ICAM-1. Efficiency of siRNA-mediated knockdown was verified by probing the blots with GPR68; β-actin was used as a loading control. Shown are representative results of 5 independent experiments.
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cell sirna transfection a549 human lung epithelial cells  (ATCC)
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GPR68 activation or overexpression exacerbates endothelial inflammation. ( A ) Cells were treated with either 25 ng/ml LPS, 10 µM Ogerin, or both for 6 h followed by western blot analysis to detect ICAM-1 protein levels. * p < 0.05, vs. control and ** p < 0.05, versus LPS, n = 3. ( B ) Cells were transfected with wild type (WT) or inactive mutant (Mut) GPR68 plasmids for 24 h followed by stimulation with 50 ng/ml of LPS for 1 or 6 h. Protein expression of phospho-NFkB, VCAM-1, and ICAM-1 were analyzed by western blotting. Probing for GFP was used as a <t>transfection</t> control. * p < 0.05, versus WT, n = 3 ( C ) Cells were transfected with empty vector (EV) or GPR68 wild type (WT) plasmids for 24 h and exposed to 50 ng/ml of LPS for 3 h. qPCR was performed to determine mRNA expression levels of TNF-α (upper panel), IL-6, IL-1β (middle panel), and IL-8, E-selectin (lower panel). * p < 0.05, versus control and ** p < 0.05, WT versus EV, n = 3. ( C ) Cells were transfected with GPR68 WT or inactive mutant (GPR Mut) plasmids for 24 h followed by stimulation with indicated dosed of LPS (3 h). mRNA levels of TNF-α (upper panel) or IL-6, IL-8, IL-1β, E-selectin, and CXCL5 (lower panel) were determined by real-time PCR. * p < 0.05, versus WT, n = 4. (E) Cells were subjected to non-specific (NS) or GPR68-targeting (siGPR68) siRNA transfection for 72 h and then stimulated with LPS for 6 h. Western blot analysis was carried out to measure the protein levels of phospho-NFkB, VCAM-1, and ICAM-1. Efficiency of siRNA-mediated knockdown was verified by probing the blots with GPR68; β-actin was used as a loading control. Shown are representative results of 5 independent experiments.
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GPR68 activation or overexpression exacerbates endothelial inflammation. ( A ) Cells were treated with either 25 ng/ml LPS, 10 µM Ogerin, or both for 6 h followed by western blot analysis to detect ICAM-1 protein levels. * p < 0.05, vs. control and ** p < 0.05, versus LPS, n = 3. ( B ) Cells were transfected with wild type (WT) or inactive mutant (Mut) GPR68 plasmids for 24 h followed by stimulation with 50 ng/ml of LPS for 1 or 6 h. Protein expression of phospho-NFkB, VCAM-1, and ICAM-1 were analyzed by western blotting. Probing for GFP was used as a transfection control. * p < 0.05, versus WT, n = 3 ( C ) Cells were transfected with empty vector (EV) or GPR68 wild type (WT) plasmids for 24 h and exposed to 50 ng/ml of LPS for 3 h. qPCR was performed to determine mRNA expression levels of TNF-α (upper panel), IL-6, IL-1β (middle panel), and IL-8, E-selectin (lower panel). * p < 0.05, versus control and ** p < 0.05, WT versus EV, n = 3. ( C ) Cells were transfected with GPR68 WT or inactive mutant (GPR Mut) plasmids for 24 h followed by stimulation with indicated dosed of LPS (3 h). mRNA levels of TNF-α (upper panel) or IL-6, IL-8, IL-1β, E-selectin, and CXCL5 (lower panel) were determined by real-time PCR. * p < 0.05, versus WT, n = 4. (E) Cells were subjected to non-specific (NS) or GPR68-targeting (siGPR68) siRNA transfection for 72 h and then stimulated with LPS for 6 h. Western blot analysis was carried out to measure the protein levels of phospho-NFkB, VCAM-1, and ICAM-1. Efficiency of siRNA-mediated knockdown was verified by probing the blots with GPR68; β-actin was used as a loading control. Shown are representative results of 5 independent experiments.

Journal: Scientific Reports

Article Title: Novel small molecule inhibitor of GPR68 attenuates endothelial dysfunction and lung injury caused by bacterial lipopolysaccharide

doi: 10.1038/s41598-025-02582-y

Figure Lengend Snippet: GPR68 activation or overexpression exacerbates endothelial inflammation. ( A ) Cells were treated with either 25 ng/ml LPS, 10 µM Ogerin, or both for 6 h followed by western blot analysis to detect ICAM-1 protein levels. * p < 0.05, vs. control and ** p < 0.05, versus LPS, n = 3. ( B ) Cells were transfected with wild type (WT) or inactive mutant (Mut) GPR68 plasmids for 24 h followed by stimulation with 50 ng/ml of LPS for 1 or 6 h. Protein expression of phospho-NFkB, VCAM-1, and ICAM-1 were analyzed by western blotting. Probing for GFP was used as a transfection control. * p < 0.05, versus WT, n = 3 ( C ) Cells were transfected with empty vector (EV) or GPR68 wild type (WT) plasmids for 24 h and exposed to 50 ng/ml of LPS for 3 h. qPCR was performed to determine mRNA expression levels of TNF-α (upper panel), IL-6, IL-1β (middle panel), and IL-8, E-selectin (lower panel). * p < 0.05, versus control and ** p < 0.05, WT versus EV, n = 3. ( C ) Cells were transfected with GPR68 WT or inactive mutant (GPR Mut) plasmids for 24 h followed by stimulation with indicated dosed of LPS (3 h). mRNA levels of TNF-α (upper panel) or IL-6, IL-8, IL-1β, E-selectin, and CXCL5 (lower panel) were determined by real-time PCR. * p < 0.05, versus WT, n = 4. (E) Cells were subjected to non-specific (NS) or GPR68-targeting (siGPR68) siRNA transfection for 72 h and then stimulated with LPS for 6 h. Western blot analysis was carried out to measure the protein levels of phospho-NFkB, VCAM-1, and ICAM-1. Efficiency of siRNA-mediated knockdown was verified by probing the blots with GPR68; β-actin was used as a loading control. Shown are representative results of 5 independent experiments.

Article Snippet: Ogerin was from Sigma (St. Louis, MO), and GPR68 antibody and DNA/siRNA transfections reagents were purchased from Thermo Fisher Scientific (Waltham, MA).

Techniques: Activation Assay, Over Expression, Western Blot, Control, Transfection, Mutagenesis, Expressing, Plasmid Preparation, Real-time Polymerase Chain Reaction, Knockdown