Journal: iScience

Article Title: Type 2 diabetes is associated with increased circulating levels of 3-hydroxydecanoate activating GPR84 and neutrophil migration

doi: 10.1016/j.isci.2022.105683

Figure Lengend Snippet:

Article Snippet: Receptor plasmid, human HCA2 (GPR109A) , Origene , N/A.

Techniques: RNAscope, Recombinant, Enzyme-linked Immunosorbent Assay, Isolation, Reporter Gene Assay, Plasmid Preparation, Software

Journal: Blood Advances

Article Title: HCAR2 is a novel receptor for heme ∗

doi: 10.1182/bloodadvances.2025016197

Figure Lengend Snippet: Functional validation of HCAR2 as a receptor for heme measured by receptor signaling. (A) Titration of heme in the activity assay in agonist mode of HCAR2/GPR109A. (B) Titration of nicotinic acid (niacin) in the activity assay in agonist mode of HCAR2/GPR109A.

Article Snippet: Cells were harvested and washed with PBS 1× and further stained with live-dead (Invitrogen; L10119A) and anti-human HCAR2 primary antibody (Novus; NBP1-92180) and labeled with chicken anti-rabbit AF647 secondary antibody (Invitrogen; A21443).

Techniques: Functional Assay, Biomarker Discovery, Titration, Activity Assay

Journal: Blood Advances

Article Title: HCAR2 is a novel receptor for heme ∗

doi: 10.1182/bloodadvances.2025016197

Figure Lengend Snippet: Biochemical validation of HCAR2 as a receptor for heme, illustrated by direct ligand/receptor binding. (A) Comparison by SPR of the interaction of immobilized recombinant HCAR2 compared to C3b (negative control) with different concentrations of heme (39 to 2500 nM). (B) Analysis of HCAR2-heme interaction by spectroscopy (0.35 to 10.2 μM). (C) Absorbance at the Soret peak (414 nM) as a function of the molar excess of heme over the protein. RU, response units.

Article Snippet: Cells were harvested and washed with PBS 1× and further stained with live-dead (Invitrogen; L10119A) and anti-human HCAR2 primary antibody (Novus; NBP1-92180) and labeled with chicken anti-rabbit AF647 secondary antibody (Invitrogen; A21443).

Techniques: Biomarker Discovery, Binding Assay, Comparison, Recombinant, Negative Control, Spectroscopy

Journal: Blood Advances

Article Title: HCAR2 is a novel receptor for heme ∗

doi: 10.1182/bloodadvances.2025016197

Figure Lengend Snippet: HCAR2 is overexpressed in hemolytic and SCD mice and its expression is regulated by heme/HO-1 axis. (A) Schematic overview of the experiment performed on mice. (B) Heat map of the normalized counts of the 3 main Hcar2 transcription factors of PHZ vs PBS (upper) and HbSS vs HbAA mice (lower). Normalized counts in each mouse are represented with row scale normalization. (C-F) Spearman correlation of Hcar2 and Hmox1 normalized gene expression levels in PHZ- and PBS-treated mice from RNAseq (C) and QuantiGene data (E) and HbSS and HbAA mice from RNAseq (D) and QuantiGene data (F). (G) QuantiGene analysis of Hcar2 gene expression levels in PHZ- and PBS-treated mice. (H) QuantiGene analysis of Hcar2 gene expression levels in HbSS and HbAA mice, either treated with PBS (left) or pretreated with SnMP, followed by an injection of 24-μM heme (right). (I-J) QuantiGene analysis of Hcar2 expression in HbAA mice injected with heme. (I) Level of expression of Hcar2 . (J) Spearman correlation of Hcar2 and Hmox1 normalized gene expression in the HbAA mice injected with PBS or heme. ∗∗∗∗ P < .0001; ∗ P < .05; Mann-Whitney test in panels G,I. ∗ P < .05; Kruskal-Wallis with Dunn test for multiple pairwise comparisons in panel H.

Article Snippet: Cells were harvested and washed with PBS 1× and further stained with live-dead (Invitrogen; L10119A) and anti-human HCAR2 primary antibody (Novus; NBP1-92180) and labeled with chicken anti-rabbit AF647 secondary antibody (Invitrogen; A21443).

Techniques: Expressing, Gene Expression, Injection, MANN-WHITNEY

Journal: Cell Reports Medicine

Article Title: Dietary vitamin B3 supplementation induces the antitumor immunity against liver cancer via biased GPR109A signaling in myeloid cell

doi: 10.1016/j.xcrm.2024.101718

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Article Snippet: HM74A APC , R&D Systems , Cat#FAB2760A; RRID: AB_573115.

Techniques: Control, Staining, Recombinant, Liposomes, Cell Isolation, CCK-8 Assay, TUNEL Assay, Apoptosis Assay, Activation Assay, Software

Journal: JBMR Plus

Article Title: 3‐(3‐Hydroxyphenyl)‐Propionic Acid (PPA) Suppresses Osteoblastic Cell Senescence to Promote Bone Accretion in Mice

doi: 10.1002/jbm4.10201

Figure Lengend Snippet: HA and PPA stimulate 35 S‐GTPγS binding to membranes from mouse osteoblastic calvarial cells transfected with GPR109A but not GPR109B. Ligand‐induced 35 S‐GTPγS binding was studied using membranes prepared from mouse fetal calvarial cells expressing mouse GPR109A ( A ) or GPR109B ( B ). Binding of 35 S‐GTPγS was determined in the presence of increased concentration of nicotinic acid, acifran, HA, or PPA. HA = hippuric acid; PPA = 3‐(3‐hydroxyphenyl)‐propionic acid.

Article Snippet: Ligand‐induced 35 S‐GTPγS binding was studied using membrane fractions prepared from mouse fetal calvarial cells stably transfected with mouse GPR109A overexpression plasmid (OriGene, Rockville, MD, USA; catalog number RG206527) or GPR109B (OriGen Technologies; catalog number RG208523).

Techniques: Binding Assay, Transfection, Expressing, Concentration Assay

Journal: Journal of cellular physiology

Article Title: Hippuric acid and 3-(3-hydroxyphenyl) propionic acid inhibit murine osteoclastogenesis through RANKL-RANK independent pathway.

doi: 10.1002/jcp.28998

Figure Lengend Snippet: FIGURE 7 HA and 3‐3‐PPA change intracellular cAMP levels in RAW264.7 cells. RAW 264.7 cells were treated with RANKL (50 ng/ml), HA (1×) and the combination of HA and RANKL, control = PBS for 4 indicated days. (a) Intracellular cAMP levels from cell lysates were measured using an ELISA method. (b) cAMP levels in cell culture medium were measured using an ELISA method. (c) Western blots showing GPR109A expression in RAW 264.7 cells were transfected with GPR109A overexpression plasmid (three different concentrations, 1× equal to 0.4 pmol/µl of DNA as suggested by manufactory), or shRNA GPR109A (three different concentrations, 1× equal to 12.5 ng/µl of RNA as suggested by manufactory), or control vector. β‐actin and Amido Black showing lording control. (d) Intracellular cAMP levels after GPR109A overexpression plasmid or shRNA GPR109A. (e) Working hypothesis of HA or 3‐3‐PPA inhibits GPR109A expression leads to cAMP accumulation in the cytoplasm to interfere osteoclastogenic signaling. *,#Significant differences versus control by t test. Data are means ± standard error with triplicates, time‐dependent changes were assessed using Cruzickʼs nonparametric test for trend. cAMP, cyclic adenosine monophosphate; HA, hippuric acid; IL, interleukin; RANKL, receptor activator of nuclear factor κ‐Β ligand; shRNA, short hairpin RNA; TNF, tumor necrosis factor [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Cells were transfected with either overexpression plasmid GPR109A (#MC211930; HCAR2, Origene), or shRNA GPR109A (#TR511005; HCAR2‐sh, Origene), or control vector (PCMV6KN, Origene) using Lipofec- tamine 3000.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Cell Culture, Western Blot, Expressing, Transfection, Over Expression, Plasmid Preparation, shRNA

Journal: Renal Failure

Article Title: Oral Astragalus polysaccharide alleviates adenine-induced kidney injury by regulating gut microbiota–short-chain fatty acids–kidney G protein-coupled receptors axis

doi: 10.1080/0886022x.2024.2429693

Figure Lengend Snippet: Figure 6. APS supplementing increase kidney GRP expression. (A) Kidney GPR41 immunohistochemical staining and analysis (×100 and ×200). (B) Kidney GPR43 immunohistochemical staining and analysis (×100 and ×200). (C) Kidney GPR109a immunohistochemical staining and analysis (×100 and ×200). (D) Western-blot analysis of GPR41, 43, and 109a in kidney tissue. The data are presented as the mean ± standard deviation of the mean for each mea surement (*p < .05, **p < .01).

Article Snippet: Sections were incubated with anti-G protein-coupled receptor 41 (GPR41) primary antibody (1:100, Lot: 2440.Pb1.AP, NOVUS, Centennial, CO), anti-GPR43 primary antibody (1:50, Lot: #617A024, Absin, Shanghai, China), and anti-GPR109a primary antibody (1:100, Lot: E105361, NOVUS, Centennial, CO) at 4 °C overnight.

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot, Standard Deviation