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recombinant adenovirus for gpr4 (BioFocus DPI)

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BioFocus DPI recombinant adenovirus for gpr4

HEK293 cells were transiently transfected with <t>GPR4</t> (A and B), OGR1 (C), TDAG8 (D), or G2A (E) plasmid, together with pRL-TK and pSRE-luc. The cells expressing the respective receptor and luciferase genes were then incubated for 6 h at the indicated pH to measure SRE promoter activity in the presence or absence of test compounds. The concentration of test compounds was as follows; 0.1 to 10 μM for compound 1 (C1) and 10 μM for compound 2 (C2) and compound 3 (C3) in (A), the indicated concentration in (B), and 10 μM for all the test compounds in (C to E). SRE activity was estimated as the ratio of firefly (pSRE-luc) and Renilla (pRL-TK) luciferase activities and the results are shown as percentages of the maximal activity (ratio) obtained at optimum pH for each receptor in (A and C-E): the maximal activity was 12.1 ± 3.8 for GPR4 at pH 6.8 in (A), 21.4 ± 7.1 for OGR1 at pH 6.4 in (C), 36.4 ± 0.4 for TDAG8 at pH 6.6 in (D), and 18.6 ± 6.7 for G2A at pH 6.4 in (E). In (B), the activity was expressed as percentages of the value obtained at pH 7.0. Results are means ± SEM of 4 to 6 determinations from two to three separate experiments. In (A), effect of each test compound is significantly different from control (* p < 0.05), while effects of all the test compounds at pH 7.2, 7.0, and 6.8 are significantly different from control (# p < 0.05). In (B), effect of C2 or C3 is significantly different from that of C1 (¶ p < 0.05). In (C to E), effect of each test compound is significantly different from control (* p < 0.05).

Recombinant Adenovirus For Gpr4, supplied by BioFocus DPI, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/gpr4/pmc04466532-77-4-8?v=BioFocus+DPI
Average 90 stars, based on 1 article reviews

recombinant adenovirus for gpr4 - by Bioz Stars, 2026-08

90/100 stars

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1) Product Images from "Characterization of Imidazopyridine Compounds as Negative Allosteric Modulators of Proton-Sensing GPR4 in Extracellular Acidification-Induced Responses"

Article Title: Characterization of Imidazopyridine Compounds as Negative Allosteric Modulators of Proton-Sensing GPR4 in Extracellular Acidification-Induced Responses

Journal: PLoS ONE

doi: 10.1371/journal.pone.0129334

HEK293 cells were transiently transfected with GPR4 (A and B), OGR1 (C), TDAG8 (D), or G2A (E) plasmid, together with pRL-TK and pSRE-luc. The cells expressing the respective receptor and luciferase genes were then incubated for 6 h at the indicated pH to measure SRE promoter activity in the presence or absence of test compounds. The concentration of test compounds was as follows; 0.1 to 10 μM for compound 1 (C1) and 10 μM for compound 2 (C2) and compound 3 (C3) in (A), the indicated concentration in (B), and 10 μM for all the test compounds in (C to E). SRE activity was estimated as the ratio of firefly (pSRE-luc) and Renilla (pRL-TK) luciferase activities and the results are shown as percentages of the maximal activity (ratio) obtained at optimum pH for each receptor in (A and C-E): the maximal activity was 12.1 ± 3.8 for GPR4 at pH 6.8 in (A), 21.4 ± 7.1 for OGR1 at pH 6.4 in (C), 36.4 ± 0.4 for TDAG8 at pH 6.6 in (D), and 18.6 ± 6.7 for G2A at pH 6.4 in (E). In (B), the activity was expressed as percentages of the value obtained at pH 7.0. Results are means ± SEM of 4 to 6 determinations from two to three separate experiments. In (A), effect of each test compound is significantly different from control (* p < 0.05), while effects of all the test compounds at pH 7.2, 7.0, and 6.8 are significantly different from control (# p < 0.05). In (B), effect of C2 or C3 is significantly different from that of C1 (¶ p < 0.05). In (C to E), effect of each test compound is significantly different from control (* p < 0.05).

Figure Legend Snippet: HEK293 cells were transiently transfected with GPR4 (A and B), OGR1 (C), TDAG8 (D), or G2A (E) plasmid, together with pRL-TK and pSRE-luc. The cells expressing the respective receptor and luciferase genes were then incubated for 6 h at the indicated pH to measure SRE promoter activity in the presence or absence of test compounds. The concentration of test compounds was as follows; 0.1 to 10 μM for compound 1 (C1) and 10 μM for compound 2 (C2) and compound 3 (C3) in (A), the indicated concentration in (B), and 10 μM for all the test compounds in (C to E). SRE activity was estimated as the ratio of firefly (pSRE-luc) and Renilla (pRL-TK) luciferase activities and the results are shown as percentages of the maximal activity (ratio) obtained at optimum pH for each receptor in (A and C-E): the maximal activity was 12.1 ± 3.8 for GPR4 at pH 6.8 in (A), 21.4 ± 7.1 for OGR1 at pH 6.4 in (C), 36.4 ± 0.4 for TDAG8 at pH 6.6 in (D), and 18.6 ± 6.7 for G2A at pH 6.4 in (E). In (B), the activity was expressed as percentages of the value obtained at pH 7.0. Results are means ± SEM of 4 to 6 determinations from two to three separate experiments. In (A), effect of each test compound is significantly different from control (* p < 0.05), while effects of all the test compounds at pH 7.2, 7.0, and 6.8 are significantly different from control (# p < 0.05). In (B), effect of C2 or C3 is significantly different from that of C1 (¶ p < 0.05). In (C to E), effect of each test compound is significantly different from control (* p < 0.05).

Techniques Used: Transfection, Plasmid Preparation, Expressing, Luciferase, Incubation, Activity Assay, Concentration Assay, Control

GPR4-expressing CHO cells (A), GPR4-expressing COS7 cells (B), or TDAG8-expressing COS7 cells (C) were incubated for 30 min to measure cAMP accumulation under the indicated pH with or without 1 μM compound 1 (C1) and/or 1 μM forskolin (Fors). Results are expressed as means ± SD of three determinations of the representative experiment in (A) and expressed as means ± SEM of five determinations of three separate experiments in (B and C). The effect of compound 1 was significant (* p < 0.05). HUVECs infected with GPR4 adenovirus were incubated for 6 h in the presence of the indicated concentrations of compound 1 in (D and E) or in the presence of absence of 1 μM compound 1 in (F and G) at the indicated pH to measure mRNAs for VCAM-1 (D), ICAM-1 (E), CXCL2 (F), and IL-8 (G). The mRNA expression normalized to GAPDH was expressed as percentages of the value in the absence of compound 1 at pH 6.8. These values at pH 6.8 were 22.7 ± 6.6 for VCAM1 mRNA, 15.1 ± 4.1 for ICAM1 mRNA, 226 ± 20 for CXCL2 mRNA, 12.9 ± 0.9 for IL-8 mRNA (normalized to GAPDH x 10 3 ). The results are means ± SEM of three to five separate experiments. The effect of compound 1 was significant (* p < 0.05). (H) AoSMCs harvested from 10-cm dish were prelabeled with fura-2/AM. The cells were first incubated with compound 1 (1 μM) and then further incubated under indicated pH with or without LPA (1 μM) and SPC (20 μM) to monitor [Ca 2+ ] i . The net [Ca 2+ ] i change (peak value-basal value) at around 15 s was calculated. Data are means ± SEM from three separate experiments.

Figure Legend Snippet: GPR4-expressing CHO cells (A), GPR4-expressing COS7 cells (B), or TDAG8-expressing COS7 cells (C) were incubated for 30 min to measure cAMP accumulation under the indicated pH with or without 1 μM compound 1 (C1) and/or 1 μM forskolin (Fors). Results are expressed as means ± SD of three determinations of the representative experiment in (A) and expressed as means ± SEM of five determinations of three separate experiments in (B and C). The effect of compound 1 was significant (* p < 0.05). HUVECs infected with GPR4 adenovirus were incubated for 6 h in the presence of the indicated concentrations of compound 1 in (D and E) or in the presence of absence of 1 μM compound 1 in (F and G) at the indicated pH to measure mRNAs for VCAM-1 (D), ICAM-1 (E), CXCL2 (F), and IL-8 (G). The mRNA expression normalized to GAPDH was expressed as percentages of the value in the absence of compound 1 at pH 6.8. These values at pH 6.8 were 22.7 ± 6.6 for VCAM1 mRNA, 15.1 ± 4.1 for ICAM1 mRNA, 226 ± 20 for CXCL2 mRNA, 12.9 ± 0.9 for IL-8 mRNA (normalized to GAPDH x 10 3 ). The results are means ± SEM of three to five separate experiments. The effect of compound 1 was significant (* p < 0.05). (H) AoSMCs harvested from 10-cm dish were prelabeled with fura-2/AM. The cells were first incubated with compound 1 (1 μM) and then further incubated under indicated pH with or without LPA (1 μM) and SPC (20 μM) to monitor [Ca 2+ ] i . The net [Ca 2+ ] i change (peak value-basal value) at around 15 s was calculated. Data are means ± SEM from three separate experiments.

Techniques Used: Expressing, Incubation, Infection

Permanent cell line expressing GFP-conjugated GPR4 or V 1a vasopressin receptor was incubated for 1 h with or without 1 μM [Arg 8 ]-vasopressin (AVP) or 1 μM compound 1 (C1) under the indicated pH to monitor of receptor internalization. After fixation, nucleus was stained with DAPI (blue) and localization of GFP-receptor (green) was monitored. Scale bar: 20 μm. The results are representative of three to four separate experiments. The higher magnification image of the small square is shown in the large square.

Figure Legend Snippet: Permanent cell line expressing GFP-conjugated GPR4 or V 1a vasopressin receptor was incubated for 1 h with or without 1 μM [Arg 8 ]-vasopressin (AVP) or 1 μM compound 1 (C1) under the indicated pH to monitor of receptor internalization. After fixation, nucleus was stained with DAPI (blue) and localization of GFP-receptor (green) was monitored. Scale bar: 20 μm. The results are representative of three to four separate experiments. The higher magnification image of the small square is shown in the large square.

Techniques Used: Expressing, Incubation, Staining

HEK293 cells were transiently transfected with plasmid of GPR4 (A and D), OGR1 (B), TDAG8 (C), or H165F/H269F double mutant of GPR4 (GPR4-mutant; E and F), together with pRL-TK and pSRE-luc. The cells expressing the respective receptor and luciferase genes were then incubated for 6 h at the indicated pH to measure SRE promoter activity in the presence or absence of psychosine (Psy; 10 μM), N -acetyl-psychosine ( N Ac-Psy; 10 μM), or compound 1 (C1; 100 nM). SRE activity was estimated as the ratio of firefly (pSRE-luc) and Renilla (pRL-TK) luciferase activities and the results are shown as percentages of the maximal activity (ratio) obtained at optimum pH for each receptor in (A-D), as shown in . In GPR4 mutant experiments (E and F), the activity was expressed as percentages of the value obtained at pH 6.8 in wild-type GPR4 (A). Results are means ± SEM of 4 to 6 determinations from two to three separate experiments. The effect of each test compound was significantly different from control (* p < 0.05).

Figure Legend Snippet: HEK293 cells were transiently transfected with plasmid of GPR4 (A and D), OGR1 (B), TDAG8 (C), or H165F/H269F double mutant of GPR4 (GPR4-mutant; E and F), together with pRL-TK and pSRE-luc. The cells expressing the respective receptor and luciferase genes were then incubated for 6 h at the indicated pH to measure SRE promoter activity in the presence or absence of psychosine (Psy; 10 μM), N -acetyl-psychosine ( N Ac-Psy; 10 μM), or compound 1 (C1; 100 nM). SRE activity was estimated as the ratio of firefly (pSRE-luc) and Renilla (pRL-TK) luciferase activities and the results are shown as percentages of the maximal activity (ratio) obtained at optimum pH for each receptor in (A-D), as shown in . In GPR4 mutant experiments (E and F), the activity was expressed as percentages of the value obtained at pH 6.8 in wild-type GPR4 (A). Results are means ± SEM of 4 to 6 determinations from two to three separate experiments. The effect of each test compound was significantly different from control (* p < 0.05).

Techniques Used: Transfection, Plasmid Preparation, Mutagenesis, Expressing, Luciferase, Incubation, Activity Assay, Control

Extracellular protons induce GPR4 activation through histidine residues and subsequent activation of G protein/effector systems, i . e ., G s /cAMP system and G 13 /Rho system, resulting in the mRNA expression of adhesion molecules and SRE transcriptional activation, respectively. Similarly to other GPCRs, GPR4 is desensitized by its internalization in response to extracellular acidification. Both imidazopyridine compound and psychosine inhibit the proton/GPR4 function but by different action modes with respect to the histidine susceptibility. See text more detail.

Figure Legend Snippet: Extracellular protons induce GPR4 activation through histidine residues and subsequent activation of G protein/effector systems, i . e ., G s /cAMP system and G 13 /Rho system, resulting in the mRNA expression of adhesion molecules and SRE transcriptional activation, respectively. Similarly to other GPCRs, GPR4 is desensitized by its internalization in response to extracellular acidification. Both imidazopyridine compound and psychosine inhibit the proton/GPR4 function but by different action modes with respect to the histidine susceptibility. See text more detail.

Techniques Used: Activation Assay, Expressing

Image Search Results

Molecular changes following ETO administration in the retrotrapezoid nucleus (RTN), the solitary tract nucleus (NTS), and the medial hypothalamus (MH) of female and male rats. (A–C) Quantitative PCR analysis of Nmb , transcription factor Phox2b , and proton‐sensing channels, Gpr4 and Task2 , in the RTN (A), NTS (B), and MH (C) of females and males. Naive males showed higher expression levels of Nmb and Gpr4 in the RTN (A) and Phox2b , Gpr4 , and Pgr in the NTS (B) compared with females. Following lesion or ETO treatment, Nmb expression was significantly reduced in both SHAM‐ and ETO‐treated animals compared with naive controls, in males and females (A). ETO‐treated females showed upregulation of Task2 and Gpr4 mRNA levels in the RTN (A), and downregulation of Phox2b and Task2 mRNA levels in the NTS (B), consistent with the observed recovery of the CO 2 chemoreflex. No significant changes were observed in the MH (C). In males, no significant transcriptional changes were detected in the RTN, NTS, or MH, except for the reduction of Nmb in the RTN. Graphs show mean ± SD with individual values represented as 2 −ΔCt with all statistical analyses performed on ΔCt values. Comparisons between naive males and females were analyzed using unpaired t ‐tests or Mann–Whitney U tests, whereas differences among experimental groups within each sex were analyzed using one‐way ANOVA (Tukey post hoc) or Kruskal–Wallis ANOVA (Dunn's post hoc). * p < 0.05, ** p < 0.01, *** p < 0.001.

Journal: Acta Physiologica (Oxford, England)

Article Title: Sex Differences in the Effects of Etonogestrel on Respiratory Recovery in an In Vivo Rat Model of Central Chemoreflex Impairment

doi: 10.1111/apha.70194

Figure Lengend Snippet: Molecular changes following ETO administration in the retrotrapezoid nucleus (RTN), the solitary tract nucleus (NTS), and the medial hypothalamus (MH) of female and male rats. (A–C) Quantitative PCR analysis of Nmb , transcription factor Phox2b , and proton‐sensing channels, Gpr4 and Task2 , in the RTN (A), NTS (B), and MH (C) of females and males. Naive males showed higher expression levels of Nmb and Gpr4 in the RTN (A) and Phox2b , Gpr4 , and Pgr in the NTS (B) compared with females. Following lesion or ETO treatment, Nmb expression was significantly reduced in both SHAM‐ and ETO‐treated animals compared with naive controls, in males and females (A). ETO‐treated females showed upregulation of Task2 and Gpr4 mRNA levels in the RTN (A), and downregulation of Phox2b and Task2 mRNA levels in the NTS (B), consistent with the observed recovery of the CO 2 chemoreflex. No significant changes were observed in the MH (C). In males, no significant transcriptional changes were detected in the RTN, NTS, or MH, except for the reduction of Nmb in the RTN. Graphs show mean ± SD with individual values represented as 2 −ΔCt with all statistical analyses performed on ΔCt values. Comparisons between naive males and females were analyzed using unpaired t ‐tests or Mann–Whitney U tests, whereas differences among experimental groups within each sex were analyzed using one‐way ANOVA (Tukey post hoc) or Kruskal–Wallis ANOVA (Dunn's post hoc). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The assays used were: rat Phox2b (ID: Rn01413076_mH), rat Pgr (ID: Rn01448227_m1), rat Gpr4 (ID: Rn02585915), rat Task2 (ID: Rn01755927), rat Nmb (ID: Rn01478123_m1), and the endogenous control rat Actb (ID: Rn00667869_m1).

Techniques: Real-time Polymerase Chain Reaction, Expressing, MANN-WHITNEY

Treatment with GPR4 antagonist NE-52-QQ57 improves survival of SARS-CoV-2-infected K18-hACE2 mice. Mice were treated with GPR4 antagonist NE-52-QQ57 or vehicle control for up to 6 days starting from 4 dpi. (A) The survival rate of SARS-CoV-2-infected K18-hACE2 mice is increased by the administration of the GPR4 antagonist. Ten-month-old male and female K18-hACE2 transgenic mice were intranasally inoculated with 1000 PFU of SARS-CoV-2 (N = 12). Survival analysis was performed using the Kaplan-Meier method with a log-rank (Mantel-Cox) test, *p < 0.05. (B) Daily body weight changes in GPR4 antagonist-treated or vehicle control mice were recorded up to 10 dpi or until the mice reached the humane endpoint. The difference in body weight change was analyzed using multiple unpaired t-tests. (C) RT-qPCR was conducted to quantify the expression of GPR4 in non-infected (PBS) and SARS-CoV-2-infected mouse lung tissues (compared using two-tailed Mann-Whitney test) (N = 6 for PBS no virus inoculation; N = 12 for vehicle, N = 10 for GPR4 antagonist). Error bars indicate means ± SEM. **p < 0.01. (D) Representative pictures of mouse lung histology (H&E staining) with mild or severe histopathology in vehicle or GPR4 antagonist-treated mice. Scale bar = 20 µm. (E) Mouse lung histopathological score. Two-tailed Student’s t-test did not indicate significance.

Journal: Frontiers in Pharmacology

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.3389/fphar.2025.1549296

Figure Lengend Snippet: Treatment with GPR4 antagonist NE-52-QQ57 improves survival of SARS-CoV-2-infected K18-hACE2 mice. Mice were treated with GPR4 antagonist NE-52-QQ57 or vehicle control for up to 6 days starting from 4 dpi. (A) The survival rate of SARS-CoV-2-infected K18-hACE2 mice is increased by the administration of the GPR4 antagonist. Ten-month-old male and female K18-hACE2 transgenic mice were intranasally inoculated with 1000 PFU of SARS-CoV-2 (N = 12). Survival analysis was performed using the Kaplan-Meier method with a log-rank (Mantel-Cox) test, *p < 0.05. (B) Daily body weight changes in GPR4 antagonist-treated or vehicle control mice were recorded up to 10 dpi or until the mice reached the humane endpoint. The difference in body weight change was analyzed using multiple unpaired t-tests. (C) RT-qPCR was conducted to quantify the expression of GPR4 in non-infected (PBS) and SARS-CoV-2-infected mouse lung tissues (compared using two-tailed Mann-Whitney test) (N = 6 for PBS no virus inoculation; N = 12 for vehicle, N = 10 for GPR4 antagonist). Error bars indicate means ± SEM. **p < 0.01. (D) Representative pictures of mouse lung histology (H&E staining) with mild or severe histopathology in vehicle or GPR4 antagonist-treated mice. Scale bar = 20 µm. (E) Mouse lung histopathological score. Two-tailed Student’s t-test did not indicate significance.

Article Snippet: Commercial primers/probe sets specific for mouse Gpr4 (Mm01322176_s1), Il-1β (Mm00434228_m1), Il-6 (Mm00446190_m1), Il-10 (Mm01288386_m1), Il-18 (Mm00434226_m1), Tnf-α (Mm00443258_m1), Atf3 (Mm00476033_m1), Cox2/Ptgs2 (Mm00478374_m1), Cxcl2 (Mm00436450_m1), E-selectin (Mm00441278_m1), Icam1 (Mm00516023_m1), Vcam1 (Mm01320970_m1), human GPR4 (Hs00270999_s1), mouse Ace2 (Mm01159006_m1), human ACE2 (Hs01085333_m1), mouse Tmprss2 (Mm00443687_m1), and human TMPRSS2 (Hs01122322_m1) were purchased from ThermoFisher and the expressions were normalized to 18S rRNA (HS99999901_s1) levels.

Techniques: Infection, Control, Transgenic Assay, Quantitative RT-PCR, Expressing, Two Tailed Test, MANN-WHITNEY, Virus, Staining, Histopathology

GPR4 antagonist treatment reduces cytokine and chemokine levels in K18-hACE2 mice infected with SARS-CoV-2. (A) Fold change in gene expression levels of specified cytokines, chemokines, and other inflammatory genes assessed via RT-qPCR and normalized to 18S rRNA, compared with vehicle controls in mouse lung homogenates (N = 12 for vehicle, N = 10 for GPR4 antagonist). *p < 0.05. Gene expression in the lung of control mice (PBS) without SARS-CoV-2 infection was set as 1. (B) Cytokine/chemokine protein levels in mouse lung tissues measured by the Luminex multiplex platform. (C) Cytokine/chemokine protein levels in mouse serum measured by the Luminex multiplex platform. Statistical differences in cytokine/chemokine levels were analyzed using the one-tailed Mann-Whitney test (N = 12 for vehicle, N = 10 for GPR4 antagonist). Error bars indicate mean ± SEM.

Journal: Frontiers in Pharmacology

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.3389/fphar.2025.1549296

Figure Lengend Snippet: GPR4 antagonist treatment reduces cytokine and chemokine levels in K18-hACE2 mice infected with SARS-CoV-2. (A) Fold change in gene expression levels of specified cytokines, chemokines, and other inflammatory genes assessed via RT-qPCR and normalized to 18S rRNA, compared with vehicle controls in mouse lung homogenates (N = 12 for vehicle, N = 10 for GPR4 antagonist). *p < 0.05. Gene expression in the lung of control mice (PBS) without SARS-CoV-2 infection was set as 1. (B) Cytokine/chemokine protein levels in mouse lung tissues measured by the Luminex multiplex platform. (C) Cytokine/chemokine protein levels in mouse serum measured by the Luminex multiplex platform. Statistical differences in cytokine/chemokine levels were analyzed using the one-tailed Mann-Whitney test (N = 12 for vehicle, N = 10 for GPR4 antagonist). Error bars indicate mean ± SEM.

Techniques: Infection, Gene Expression, Quantitative RT-PCR, Control, Luminex, Multiplex Assay, One-tailed Test, MANN-WHITNEY

SARS-CoV-2 viral load in the lungs and brains from K18-hACE2 mice that received either GPR4 antagonist or vehicle. (A) RT-qPCR to quantify viral RNA levels in mouse lung tissues (RNA copies/μg lung RNA). The data were analyzed using the two-tailed unpaired t-test and shown in mean ± SEM (N = 12 for vehicle, N = 10 for GPR4 antagonist). (B) Plaque assays were analyzed to determine the infectious viral titers (PFU/mg lung) in the lungs of vehicle- and GPR4 antagonist-treated mice infected with SARS-CoV-2. The limit of detection (LOD = 5 PFU/mg lung) is indicated by the dotted horizontal line. *p < 0.05. (C) Analysis of SARS-CoV-2 virus nucleocapsid distribution in mouse brain through IHC. The percentage of SARS-CoV-2 positive viral staining in the mouse brain was assessed using a microscope (N = 12 for vehicle, N = 12 for GPR4 antagonist). In the vehicle group, 3 out of 12 mouse brains (3/12) are negative for SARS-CoV-2 and 9 out of 12 are positive. In comparison, 8 out of 12 are negative and 4 out of 12 mouse brains are positive for SARS-CoV-2 in the GPR4 antagonist group. Scale bar = 20 μm. Error bars indicate mean ± SEM. (D) SARS-CoV-2 positive ratio in the brains of mice treated with GPR4 antagonist or vehicle. Analyzed using the Chi-square test, *p < 0.05.

Journal: Frontiers in Pharmacology

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.3389/fphar.2025.1549296

Figure Lengend Snippet: SARS-CoV-2 viral load in the lungs and brains from K18-hACE2 mice that received either GPR4 antagonist or vehicle. (A) RT-qPCR to quantify viral RNA levels in mouse lung tissues (RNA copies/μg lung RNA). The data were analyzed using the two-tailed unpaired t-test and shown in mean ± SEM (N = 12 for vehicle, N = 10 for GPR4 antagonist). (B) Plaque assays were analyzed to determine the infectious viral titers (PFU/mg lung) in the lungs of vehicle- and GPR4 antagonist-treated mice infected with SARS-CoV-2. The limit of detection (LOD = 5 PFU/mg lung) is indicated by the dotted horizontal line. *p < 0.05. (C) Analysis of SARS-CoV-2 virus nucleocapsid distribution in mouse brain through IHC. The percentage of SARS-CoV-2 positive viral staining in the mouse brain was assessed using a microscope (N = 12 for vehicle, N = 12 for GPR4 antagonist). In the vehicle group, 3 out of 12 mouse brains (3/12) are negative for SARS-CoV-2 and 9 out of 12 are positive. In comparison, 8 out of 12 are negative and 4 out of 12 mouse brains are positive for SARS-CoV-2 in the GPR4 antagonist group. Scale bar = 20 μm. Error bars indicate mean ± SEM. (D) SARS-CoV-2 positive ratio in the brains of mice treated with GPR4 antagonist or vehicle. Analyzed using the Chi-square test, *p < 0.05.

Techniques: Quantitative RT-PCR, Two Tailed Test, Infection, Virus, Staining, Microscopy, Comparison

GPR4 antagonist treatment reduces CD4 + and CD8 + immune cell clusters in the brains of SARS-CoV-2-infected K18-hACE2 mice. (A) A representative image of a CD4 + immune cell cluster (indicated by the arrow) in the mouse brain, visualized using IHC with an antibody detecting CD4. Scale bar = 20 μm. (B) Quantification of the number of CD4 + immune cell clusters in the brain using microscopy. Analyzed using the two-tailed Student’s t-test, **p < 0.01. Error bars represent mean ± SEM. N = 12 for the vehicle group, and N = 12 for the GPR4 antagonist group. (C) A representative image of a CD8 + immune cell cluster (indicated by the arrow) in the mouse brain, visualized using IHC with an antibody detecting CD8. Scale bar = 20 μm. (D) Quantification of the number of CD8 + immune cell clusters in the brain using microscopy. Analyzed by the two-tailed Student’s t-test, *p < 0.05. Error bars represent mean ± SEM. N = 12 for the vehicle group, and N = 12 for the GPR4 antagonist group.

Journal: Frontiers in Pharmacology

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.3389/fphar.2025.1549296

Figure Lengend Snippet: GPR4 antagonist treatment reduces CD4 + and CD8 + immune cell clusters in the brains of SARS-CoV-2-infected K18-hACE2 mice. (A) A representative image of a CD4 + immune cell cluster (indicated by the arrow) in the mouse brain, visualized using IHC with an antibody detecting CD4. Scale bar = 20 μm. (B) Quantification of the number of CD4 + immune cell clusters in the brain using microscopy. Analyzed using the two-tailed Student’s t-test, **p < 0.01. Error bars represent mean ± SEM. N = 12 for the vehicle group, and N = 12 for the GPR4 antagonist group. (C) A representative image of a CD8 + immune cell cluster (indicated by the arrow) in the mouse brain, visualized using IHC with an antibody detecting CD8. Scale bar = 20 μm. (D) Quantification of the number of CD8 + immune cell clusters in the brain using microscopy. Analyzed by the two-tailed Student’s t-test, *p < 0.05. Error bars represent mean ± SEM. N = 12 for the vehicle group, and N = 12 for the GPR4 antagonist group.

Techniques: Infection, Microscopy, Two Tailed Test

Anti-SARS-CoV-2 effects of GPR4 antagonist in vitro . (A) GPR4 antagonist incubated with SARS-CoV-2 (100 PFU) for 1h before infecting Vero E6 cells. GPR4 antagonist-containing medium was removed after infection. Plaque formation was measured to determine the infectious SARS-CoV-2 viral titer. N = 3 samples. (B) Viral RNA in the SARS-CoV-2-infected Vero E6 cells (100 PFU inoculum) treated with various concentrations of GPR4 antagonist was determined 24 h post-infection. The GPR4 antagonist was maintained in the medium until cell assessment 24 h after treatment. Viral RNA isolated from Vero E6 cells was quantified by RT-qPCR targeting the nucleocapsid gene. N = 3 samples. *p < 0.05, **p < 0.01, ****p < 0.0001. (C) The infectious viral load by the plaque assay in SARS-CoV-2-infected Vero E6 cells (30 PFU inoculum) in response to treatment of vehicle DMSO or GPR4 antagonist at 72 h post-infection. The GPR4 antagonist was maintained in the medium for 72 h until cell assessment. N = 3 samples. **p < 0.01, ***p < 0.001. Comparisons between groups were analyzed by one-way ANOVA followed by post hoc Dunnett’s test. Error bars indicate mean ± SEM.

Journal: Frontiers in Pharmacology

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.3389/fphar.2025.1549296

Figure Lengend Snippet: Anti-SARS-CoV-2 effects of GPR4 antagonist in vitro . (A) GPR4 antagonist incubated with SARS-CoV-2 (100 PFU) for 1h before infecting Vero E6 cells. GPR4 antagonist-containing medium was removed after infection. Plaque formation was measured to determine the infectious SARS-CoV-2 viral titer. N = 3 samples. (B) Viral RNA in the SARS-CoV-2-infected Vero E6 cells (100 PFU inoculum) treated with various concentrations of GPR4 antagonist was determined 24 h post-infection. The GPR4 antagonist was maintained in the medium until cell assessment 24 h after treatment. Viral RNA isolated from Vero E6 cells was quantified by RT-qPCR targeting the nucleocapsid gene. N = 3 samples. *p < 0.05, **p < 0.01, ****p < 0.0001. (C) The infectious viral load by the plaque assay in SARS-CoV-2-infected Vero E6 cells (30 PFU inoculum) in response to treatment of vehicle DMSO or GPR4 antagonist at 72 h post-infection. The GPR4 antagonist was maintained in the medium for 72 h until cell assessment. N = 3 samples. **p < 0.01, ***p < 0.001. Comparisons between groups were analyzed by one-way ANOVA followed by post hoc Dunnett’s test. Error bars indicate mean ± SEM.

Techniques: In Vitro, Incubation, Infection, Isolation, Quantitative RT-PCR, Plaque Assay

Journal: Frontiers in Pharmacology

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.3389/fphar.2025.1549296

Techniques: Infection, Control, Transgenic Assay, Quantitative RT-PCR, Expressing, Two Tailed Test, MANN-WHITNEY, Virus, Staining, Histopathology

Journal: Frontiers in Pharmacology

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.3389/fphar.2025.1549296

Techniques: Infection, Gene Expression, Quantitative RT-PCR, Control, Luminex, Multiplex Assay, One-tailed Test, MANN-WHITNEY

Journal: Frontiers in Pharmacology

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.3389/fphar.2025.1549296

Techniques: Quantitative RT-PCR, Two Tailed Test, Infection, Virus, Staining, Microscopy, Comparison

Journal: Frontiers in Pharmacology

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.3389/fphar.2025.1549296

Techniques: Infection, Microscopy, Two Tailed Test

Journal: Frontiers in Pharmacology

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.3389/fphar.2025.1549296

Techniques: In Vitro, Incubation, Infection, Isolation, Quantitative RT-PCR, Plaque Assay

HKSA‐activated GPR68 mediates endothelial barrier disruption. (A) HPAECs were exposed to HKSA (5 × 10 8 particles/mL) for indicated time periods followed by qRT‐PCR analysis of GPR4, GPR65, and GPR68 mRNA expression levels. * p < .05, vs. GPR4 and GPR65, n = 3. (B) Cells were transfected with GPR68 PRESTO‐Tango plasmids for 24 h and stimulated with HKSA (5 × 10 8 particles/mL) or 10 μM Ogerin. GPR68 activity was determined by measuring luminescence signal as described in Methods, and values are presented after normalizing to the unstimulated control group. GPR68: * p < .05, vs. control, n = 4. (C) Cells were treated with 10 μM Ogerin alone or in combination with HKSA, followed by TER measurements. The bar graph shows permeability changes at 15 h of cell stimulation. * p < .05, vs. control, ** p < .05, vs. HKSA, n = 4. (D) HPAECs monolayers were transfected with non‐specific control siRNA (nsRNA) or GPR68‐specific siRNA (siGPR68) for 72 h followed by stimulation with HKSA and TER monitoring. Bar graph: Pooled data at time point 15 h * p < .05, vs. corresponding controls, n = 4. (E) HPAEC were transfected with plasmids encoding wild type (WT) or E336X GPR68 mutant (Mut). After 24 h of incubation, TER measurements of control and HKSA‐stimulated groups were performed over 25 h. Bar graph: Pooled data at 15 h of cell treatment. * p < .05, vs. control, ** p < .05, vs. HKSA, n = 5. Shown are normalized resistance values.

Journal: The FASEB Journal

Article Title: Inhibition of proton sensor GPR68 suppresses endothelial dysfunction and acute lung injury caused by Staphylococcus aureus bacterial particles

doi: 10.1096/fj.202401947R

Figure Lengend Snippet: HKSA‐activated GPR68 mediates endothelial barrier disruption. (A) HPAECs were exposed to HKSA (5 × 10 8 particles/mL) for indicated time periods followed by qRT‐PCR analysis of GPR4, GPR65, and GPR68 mRNA expression levels. * p < .05, vs. GPR4 and GPR65, n = 3. (B) Cells were transfected with GPR68 PRESTO‐Tango plasmids for 24 h and stimulated with HKSA (5 × 10 8 particles/mL) or 10 μM Ogerin. GPR68 activity was determined by measuring luminescence signal as described in Methods, and values are presented after normalizing to the unstimulated control group. GPR68: * p < .05, vs. control, n = 4. (C) Cells were treated with 10 μM Ogerin alone or in combination with HKSA, followed by TER measurements. The bar graph shows permeability changes at 15 h of cell stimulation. * p < .05, vs. control, ** p < .05, vs. HKSA, n = 4. (D) HPAECs monolayers were transfected with non‐specific control siRNA (nsRNA) or GPR68‐specific siRNA (siGPR68) for 72 h followed by stimulation with HKSA and TER monitoring. Bar graph: Pooled data at time point 15 h * p < .05, vs. corresponding controls, n = 4. (E) HPAEC were transfected with plasmids encoding wild type (WT) or E336X GPR68 mutant (Mut). After 24 h of incubation, TER measurements of control and HKSA‐stimulated groups were performed over 25 h. Bar graph: Pooled data at 15 h of cell treatment. * p < .05, vs. control, ** p < .05, vs. HKSA, n = 5. Shown are normalized resistance values.

Article Snippet: Heat‐killed Staph. aureus bacterial particles (HKSA) were purchased from InvivoGen (San Diego, CA, Catalog code: tlrl‐hksa), and GPR4 inhibitor NE 52‐QQ57 was from MedKoo Biosciences Inc. (Morrisville, NC).

Techniques: Disruption, Quantitative RT-PCR, Expressing, Transfection, Activity Assay, Control, Permeability, Cell Stimulation, Mutagenesis, Incubation

OGM‐8345 attenuates HKSA‐induced GPR68 activation and endothelial barrier disruption. (A) HPAECs were transfected with GPR68 PRESTO‐Tango plasmids for 24 h followed by the addition of HKSA for indicated time periods with or without OGM‐8345 pretreatment (3 μM, 30 min). GPR68 activity was determined by measuring luminescence signal. The data are presented after normalizing to non‐treated controls. * p < .05, vs. control, ** p < .05, vs. HKSA, n = 4. (B) HPAEC were pre‐incubated with OGM‐8345 (3 μM, 30 min), and HKSA (5 × 10 8 particles/mL) was added for 3 h. qRT‐PCR was carried out to determine the mRNA expression levels of GPR68, GPR65, and GPR4. * p < .05, vs. GPR68 controls; ** p < .05, vs. GPR68 HKSA; ND p > .05 vs. GPR65 and GPR4 controls, n = 3. (C) HPAEC monolayers were treated with HKSA with or without pre‐treatment OGM‐8345 as above. Endothelial macromolecular permeability was determined by the XPerT assay. Increased FITC fluorescence reflects an increase in endothelial permeability. Bar = 20 μm. (D) HPAEC were pre‐incubated for 30 min with OGM‐8345 (3 μM) or NE52‐QQ57 (1 μM) followed by the addition of HKSA. Endothelial permeability was monitored by TER measurements. The bar graph represents normalized pooled data at 15 h of EC stimulation. * p < .05, vs. HKSA, n = 6.

Journal: The FASEB Journal

Article Title: Inhibition of proton sensor GPR68 suppresses endothelial dysfunction and acute lung injury caused by Staphylococcus aureus bacterial particles

doi: 10.1096/fj.202401947R

Figure Lengend Snippet: OGM‐8345 attenuates HKSA‐induced GPR68 activation and endothelial barrier disruption. (A) HPAECs were transfected with GPR68 PRESTO‐Tango plasmids for 24 h followed by the addition of HKSA for indicated time periods with or without OGM‐8345 pretreatment (3 μM, 30 min). GPR68 activity was determined by measuring luminescence signal. The data are presented after normalizing to non‐treated controls. * p < .05, vs. control, ** p < .05, vs. HKSA, n = 4. (B) HPAEC were pre‐incubated with OGM‐8345 (3 μM, 30 min), and HKSA (5 × 10 8 particles/mL) was added for 3 h. qRT‐PCR was carried out to determine the mRNA expression levels of GPR68, GPR65, and GPR4. * p < .05, vs. GPR68 controls; ** p < .05, vs. GPR68 HKSA; ND p > .05 vs. GPR65 and GPR4 controls, n = 3. (C) HPAEC monolayers were treated with HKSA with or without pre‐treatment OGM‐8345 as above. Endothelial macromolecular permeability was determined by the XPerT assay. Increased FITC fluorescence reflects an increase in endothelial permeability. Bar = 20 μm. (D) HPAEC were pre‐incubated for 30 min with OGM‐8345 (3 μM) or NE52‐QQ57 (1 μM) followed by the addition of HKSA. Endothelial permeability was monitored by TER measurements. The bar graph represents normalized pooled data at 15 h of EC stimulation. * p < .05, vs. HKSA, n = 6.

Techniques: Activation Assay, Disruption, Transfection, Activity Assay, Control, Incubation, Quantitative RT-PCR, Expressing, Permeability, Fluorescence

OGM‐8345 attenuates HKSA and acidosis‐induced endothelial dysfunction. (A) HPAEC were pretreated with 3 μM OGM‐8345 at both normal and acidic pH (30 min) and stimulated with HKSA for 6 h. XPerT assay was performed to evaluate EC monolayer permeability for macromolecules. FITC fluorescence signal reflects an increase in endothelial permeability. Bar = 20 μm. (B) HPAEC were exposed to HKSA at indicated pH media with or without OGM‐8345 (3 μM) or NE52‐QQ57 (1 μM) pretreatment followed by qRT‐PCR analysis of mRNA expression of selected pro‐inflammatory marker genes. * p < .05, vs. corresponding controls, ** p < .05, vs. pH 6.5 + HKSA, n = 3. (C) Cells were challenged with HKSA at pH 6.5 or pH 7.4 in the presence or absence of OGM‐8345 for 6 h, followed by western blot analysis of ICAM‐1 protein levels; β‐tubulin was used as an internal loading control. Bar graph: Normalized densitometry data; * p < .05, vs. HKSA pH 7.4; ** p < .05, vs. pH 6.5 + HKSA, n = 3.

Journal: The FASEB Journal

Article Title: Inhibition of proton sensor GPR68 suppresses endothelial dysfunction and acute lung injury caused by Staphylococcus aureus bacterial particles

doi: 10.1096/fj.202401947R

Figure Lengend Snippet: OGM‐8345 attenuates HKSA and acidosis‐induced endothelial dysfunction. (A) HPAEC were pretreated with 3 μM OGM‐8345 at both normal and acidic pH (30 min) and stimulated with HKSA for 6 h. XPerT assay was performed to evaluate EC monolayer permeability for macromolecules. FITC fluorescence signal reflects an increase in endothelial permeability. Bar = 20 μm. (B) HPAEC were exposed to HKSA at indicated pH media with or without OGM‐8345 (3 μM) or NE52‐QQ57 (1 μM) pretreatment followed by qRT‐PCR analysis of mRNA expression of selected pro‐inflammatory marker genes. * p < .05, vs. corresponding controls, ** p < .05, vs. pH 6.5 + HKSA, n = 3. (C) Cells were challenged with HKSA at pH 6.5 or pH 7.4 in the presence or absence of OGM‐8345 for 6 h, followed by western blot analysis of ICAM‐1 protein levels; β‐tubulin was used as an internal loading control. Bar graph: Normalized densitometry data; * p < .05, vs. HKSA pH 7.4; ** p < .05, vs. pH 6.5 + HKSA, n = 3.

Techniques: Permeability, Fluorescence, Quantitative RT-PCR, Expressing, Marker, Western Blot, Control

OGM‐8345 rescues HKSA‐induced lung injury in vivo. C57BL/6 mice were exposed to HKSA (2 × 10 8 bacterial particles/mouse, intranasal) for 18 h. (A, B) Total RNA was extracted from various tissues of control or HKSA‐challenged mice, and qRT‐PCR was used to measure mRNA expression levels of GPR4 and GPR68. * p < .05, vs. control, n = 3. (C–F) C57BL/6 mice were injected with OGM‐8345 (20 mg/kg, i.p.) immediately followed by intranasal administration of HKSA. BAL fluid was collected to analyze total PMNs (C) and protein content (D). * p < .05, vs. HKSA alone, n = 8. (E) Total RNA was extracted from lung tissues, and qRT‐PCR was carried out to determine mRNA expression of indicated pro‐inflammatory genes. p < .05, vs. HKSA alone, n = 6. (F) HKSA‐induced Evans blue accumulation in the lung reflects lung vascular hyperpermeability and the protective effect of OGM‐8345. Shown are representative images of lungs from five independent experiments.

Journal: The FASEB Journal

Article Title: Inhibition of proton sensor GPR68 suppresses endothelial dysfunction and acute lung injury caused by Staphylococcus aureus bacterial particles

doi: 10.1096/fj.202401947R

Figure Lengend Snippet: OGM‐8345 rescues HKSA‐induced lung injury in vivo. C57BL/6 mice were exposed to HKSA (2 × 10 8 bacterial particles/mouse, intranasal) for 18 h. (A, B) Total RNA was extracted from various tissues of control or HKSA‐challenged mice, and qRT‐PCR was used to measure mRNA expression levels of GPR4 and GPR68. * p < .05, vs. control, n = 3. (C–F) C57BL/6 mice were injected with OGM‐8345 (20 mg/kg, i.p.) immediately followed by intranasal administration of HKSA. BAL fluid was collected to analyze total PMNs (C) and protein content (D). * p < .05, vs. HKSA alone, n = 8. (E) Total RNA was extracted from lung tissues, and qRT‐PCR was carried out to determine mRNA expression of indicated pro‐inflammatory genes. p < .05, vs. HKSA alone, n = 6. (F) HKSA‐induced Evans blue accumulation in the lung reflects lung vascular hyperpermeability and the protective effect of OGM‐8345. Shown are representative images of lungs from five independent experiments.

Techniques: In Vivo, Control, Quantitative RT-PCR, Expressing, Injection

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1) Product Images from "Characterization of Imidazopyridine Compounds as Negative Allosteric Modulators of Proton-Sensing GPR4 in Extracellular Acidification-Induced Responses"

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