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: PLoS ONE

Article Title: Nicotinic Acid Receptor GPR109A Is Down-Regulated in Human Macrophage-Derived Foam Cells

doi: 10.1371/journal.pone.0062934

Figure Lengend Snippet: 5A GPR109A mRNA was not expressed in undifferentiated THP-1 cells but expression was seen after differentiation into basal macrophages. Treatment with NA for 24 hours caused significant down-regulation of GPR109A mRNA transcription (n = 3 per group). Foam cells induction down-regulated GPR109A mRNA transcription compared to basal macrophages (n = 3 per group). 5B Western blot showed the loss of protein expression of GPR109A in total cell lysates from foam cells compared to basal macrophages. 5C and 5D Confocal fluorescence immunocytochemistry demonstrated the expression of GPR109A on the cell surface in cultured THP-1 basal macrophages ( 5C ), and the down-regulation of GPR109A expression after foam cell induction ( 5D ).

Article Snippet: Protein bands were transferred to membrane using iBlot dry transfer system (Invitrogen, Paisley, UK), and probed with rabbit anti-human GPR109A (1∶1000, Imgenex, USA) primary antibody in PBS-Tween +5% BSA at 4°C overnight.

Techniques: Expressing, Western Blot, Fluorescence, Immunocytochemistry, Cell Culture

Journal: PLoS ONE

Article Title: Nicotinic Acid Receptor GPR109A Is Down-Regulated in Human Macrophage-Derived Foam Cells

doi: 10.1371/journal.pone.0062934

Figure Lengend Snippet: 6A Fluorescence immunohistochemistry staining with antibodies against CD68 and GPR109A. Immediately adjacent 10 µm cryosection was stained with Oil Red O for lipid distribution and visualized using Texas Red excitation filter (540–580 nm) in epifluorescence. CD68-positive cells were seen clustering at the interface between lipid-rich region and the overlying fibrous cap. GPR109A co-expression was seen in a sub-population of these CD68-positive cells outside of the lipid-rich region (yellow). Purple dotted line represents the boundary of the lipid-rich region as seen in the image above. 6B and 6C Loss of GPR109A expression in lipid-rich region. Confocal fluorescence images of CD68-positive cells outside of lipid-rich region are shown in 6B ; and those within the lipid-rich region, likely to represent foam cells, are shown in 6C .

Article Snippet: Protein bands were transferred to membrane using iBlot dry transfer system (Invitrogen, Paisley, UK), and probed with rabbit anti-human GPR109A (1∶1000, Imgenex, USA) primary antibody in PBS-Tween +5% BSA at 4°C overnight.

Techniques: Fluorescence, Immunohistochemistry, Staining, Expressing

Journal: PLoS ONE

Article Title: Nicotinic Acid Receptor GPR109A Is Down-Regulated in Human Macrophage-Derived Foam Cells

doi: 10.1371/journal.pone.0062934

Figure Lengend Snippet: Adipophilin (perilipin 2) staining is specific to intra-cellular lipid-droplet associated protein and represents a sensitive marker of foam cells. 7A High-powered confocal image showed the outline of intra-cellular lipid droplets. 7B GPR109A is strongly co-expressed in CD68-positive plaque macrophages without extensive lipid-loading i.e. non-foam cells; whereas plaque foam cells, which showed strong co-expression of CD68 and adipophilin due to extensive lipid-loading as shown in 7C , do not co-express GPR109A.

Article Snippet: Protein bands were transferred to membrane using iBlot dry transfer system (Invitrogen, Paisley, UK), and probed with rabbit anti-human GPR109A (1∶1000, Imgenex, USA) primary antibody in PBS-Tween +5% BSA at 4°C overnight.

Techniques: Staining, Marker, Expressing