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

Figure Lengend Snippet: Figure 6. Biased b-Arrestin/GPR109A axis activation in myeloid cells is responsible for antitumor effect of VB3 (A) Myeloid cells from WT mice or RAW264.7 were treated with/without LPC-H12 CM and VB3 for 12 h. The GPR109A mRNA expression in cells was detected (n = 3). (B) Myeloid cells from WT mice or RAW264.7 were treated with LPC-H12 CM and 50 mM VB3 for 0, 3, 6, 12, and 24 h. The protein expression of GPR109A, P-P65, and P65 was detected. (C and E) Myeloid cells from WT or GPR109A/ mice were treated with/without Hepa1-6 CM and VB3 (50 mM) for 12 h. The mRNA expression of GPR109A, Arg-1, IL-10, VEGF, CCL2, CSF-1, CXCL1, CXCL2, and CXCL5 was explored (C, n = 3). The protein expression of GPR109A, P-P65, and P65 was detected (E). (D and F) Myeloid cells from WT mice were treated with MPN (50 mM) previously for 4 h and treated with Hepa1-6 CM and VB3 (50 mM) for another 12 h. The mRNA expression of GPR109A, Arg-1, IL-10, VEGF, CCL2, CSF-1, CXCL1, CXCL2, and CXCL5 was detected (D, n = 3). The protein expression of GPR109A, P-P65, and P65 was evaluated (F). (G–I) Scheme of myeloid cells treatment and coincubation. Myeloid cells from WT mice were previously stimulated with CM for 12 h before treatment with VB3 (50 mM), ACTD (50 mM), and CHX (50 mM) for another 12 h (G). The mRNA expression (H, n = 3) and protein expression (I) of GPR109A were detected. (J) Myeloid cells from WT mice were treated with LPC-H12 CM and VB3 for 12 h. The binding between GPR109A and b-Arrestin1or b-Arrestin2 was determined. (K and L) b-Arrestin1/2-knockdown RAW264.7 cells were treated with LPC-H12 CM and VB3 (50 mM) for 12 h. The mRNA expression of GPR109A, Arg-1, IL-10, VEGF, CCL2, CSF-1, CXCL1, CXCL2, and CXCL5 was detected (K). The protein expression of GPR109A, P-P65, and P65 was explored (L). Data are presented as means ± SEM. Experiments were conducted independently in triplicates or more. Statistical analysis was performed by one-way ANOVA. ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Anti-b-arrestin2 antibody Santa Cruz Cat#sc-365445; RRID: AB_10841589 Anti-b-arrestin2 antibody Santa Cruz Cat#sc-13140; RRID: AB_626701 Anti-CD8a antibody Servicebio Cat# GB11068; RRID: AB_2905511 Anti-INOS antibody Abcam Cat# ab15323; RRID: AB_301857 Anti-AFP antibody Proteintech Cat#14550-1-AP; RRID: AB_2223933 Anti-GPR109A antibody Novus Cat# NBP1-92180; RRID: AB_11039155 Anti-GPR109A antibody Cohesion Bio Cat# CPA6195; RRID: AB_3096221 Anti-Ly6G antibody CST Cat# 87048; RRID: AB_2909808 Anti-Arg-1 antibody CST Cat# 93668; RRID: AB_2800207 Anti-Ki67 antibody CST Cat#12202; RRID: AB_2620142 Anti-Cleaved Caspase-3 Antibody CST Cat#9661; RRID: AB_2341188 Anti-Phospho-ERK1/2 antibody CST Cat# 9101S; RRID: AB_331646 Anti-ERK1/2 antibody CST Cat# 9102S; RRID: AB_330744 Anti-b-arrestin1 antibody CST Cat#12697S; RRID: AB_2797996 Anti-NF-kB p65 antibody CST Cat# 4764; RRID: AB_823578 Anti-Phospho-NF-kB p65 antibody CST Cat# 3033; RRID: AB_331284 Anti-Phospho-PKA C antibody CST Cat#4781; RRID: AB_2300165 PKA C-a Antibody antibody CST Cat#4782; RRID: AB_2170170 Anti-GAPDH antibody CST Cat# 2118L; RRID: AB_561053 Anti-rabbit IgG HRP-linked CST Cat# 7074S; RRID: AB_2099233 Anti-mouse IgG HRP-linked CST Cat# 7076S; RRID: AB_330924 Chemicals, peptides, and recombinant proteins Mouse GM-CSF Novoprotein Cat# CK02 Mouse G-CSF Novoprotein Cat# CB53 Mouse M-CSF Novoprotein Cat# CB34 Mouse IL-2 Novoprotein Cat# CK24 Vitamin B3 Sigma Cat#N4126-100G Lenvatinib Psaitong Cat# L10232 Mepenzolate bromide Sigma Cat# M5651 Actinomycin D MCE Cat#HY-17559 MK-0354 MCE Cat#HY-13008 (Continued on next page) Cell Reports Medicine 5, 101718, September 17, 2024 e3

Techniques: Activation Assay, Expressing, Binding Assay, Knockdown

Journal: Cell metabolism

Article Title: Chlamydia pneumoniae hijacks a host auto-regulatory IL-1β loop to drive foam cell formation and accelerate atherosclerosis

doi: 10.1016/j.cmet.2018.05.027

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-Gpr109a , Biorbyt , Cat # orb13445.

Techniques: Virus, Recombinant, Transfection, Cholesterol Assay, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Membrane, Isolation, Microarray, Software

Journal: The Febs Journal

Article Title: Butyrate suppresses mucosal inflammation in inflammatory bowel disease primarily through HDAC3 inhibition in monocytes and macrophages

doi: 10.1111/febs.70289

Figure Lengend Snippet: MCT1, GPR109A, and HDAC3 expression in intestinal mucosa of healthy controls and inflammatory bowel disease patients. (A) Gene expression of SLC16A1 , HCAR2 , and HDAC3 from bulk RNA‐sequencing data in colon biopsies of healthy controls (HC n = 48), non‐inflamed (NI) and inflamed (I) mucosa from patients with Crohn's disease (CD; NI = 162, I = 73), and ulcerative colitis (UC; NI = 145 and I = 121). The Y ‐axis in the graphs represents normalized gene counts, obtained through trimmed mean of M ‐values normalization method of raw counts from high‐throughput sequencing. These normalized values are used to depict relative gene expression levels, enabling comparison across samples. Data are presented as medians and interquartile ranges. Statistical analysis: Kruskal–Wallis test. * P < 0.05; ** P < 0.01, *** P < 0.001; **** P < 0.0001. (B) Representative immunofluorescence images and quantification of MCT1, GPR109A and HDAC3 distribution in the epithelium and lamina propria (LP) (image resolution: 0.50 μm per pixel). Positive immunoreactivity is indicated by white arrows for colonic epithelium and white arrowheads for lamina propria (LP) mononuclear cells, evaluated in mucosa areas from HC ( n = 3), and NI and I mucosa from CD and UC patients ( n = 3 per group) from the Chilean research center. Images for MCT1 and GPR109A were zoomed from a 20× magnification, while images for HDAC3, were zoomed from a 40× magnification (scale bar: 50 μm).

Article Snippet: Tissue and organoid samples were blocked with PBS 1× containing 1% BSA for 30 min at room temperature and incubated overnight at 4 °C with the following primary antibodies: rabbit anti‐human HCAR2 (GPR109A) (1 : 50, Cat No HPA028660, polyclonal), rabbit anti‐human SLC16A1 (MCT1) (1 : 250 for tissue or 1 : 100 for organoids, Cat No HPA003324, polyclonal) (both from Sigma‐Aldrich, Zwijndrecht, The Netherlands), or rabbit anti‐human HDAC3 (1 : 200, Cat No 10255‐1‐AP polyclonal, Proteintech, Rosemont, IL, USA).

Techniques: Expressing, Gene Expression, RNA Sequencing, Next-Generation Sequencing, Comparison, Immunofluorescence

Journal: The Febs Journal

Article Title: Butyrate suppresses mucosal inflammation in inflammatory bowel disease primarily through HDAC3 inhibition in monocytes and macrophages

doi: 10.1111/febs.70289

Figure Lengend Snippet: SLC16A1 , HCAR2 , and HDAC3 expression in colon epithelial and immune cells. Dot plots of single‐cell RNA‐seq (scRNA‐seq) data show genes in columns and cells in rows. The dot size represents the percentage of cells expressing the gene, and the color indicates the scaled mean expression. Healthy controls (HC), Crohn's disease (CD), ulcerative colitis (UC), and non‐inflamed (NI) or inflamed (I) mucosa are indicated as specified in the datasets. ScRNA‐seq datasets for SLC16A1 , HCAR2 , and HDAC3 are shown in (A, C) for colon epithelial cells and (B, D) for colon immune cells. Data were obtained from the studies of (A, B) Kong et al . (colon tissue from CD patients and HC) and (C, D) Smillie et al . (colon tissue from UC patients and HC) .

Article Snippet: Tissue and organoid samples were blocked with PBS 1× containing 1% BSA for 30 min at room temperature and incubated overnight at 4 °C with the following primary antibodies: rabbit anti‐human HCAR2 (GPR109A) (1 : 50, Cat No HPA028660, polyclonal), rabbit anti‐human SLC16A1 (MCT1) (1 : 250 for tissue or 1 : 100 for organoids, Cat No HPA003324, polyclonal) (both from Sigma‐Aldrich, Zwijndrecht, The Netherlands), or rabbit anti‐human HDAC3 (1 : 200, Cat No 10255‐1‐AP polyclonal, Proteintech, Rosemont, IL, USA).

Techniques: Expressing, RNA Sequencing

Journal: The Febs Journal

Article Title: Butyrate suppresses mucosal inflammation in inflammatory bowel disease primarily through HDAC3 inhibition in monocytes and macrophages

doi: 10.1111/febs.70289

Figure Lengend Snippet: Regulation of SLC16A1 , HCAR2 , and HDAC3 expression by butyrate in ex vivo ‐cultured biopsies. mRNA levels of SLC16A1 (A), HCAR2 (B) and HDAC3 (C) were measured in ex vivo ‐cultured biopsies from colonic mucosa of healthy controls (HC) ( n = 3) and non‐inflamed (NI, n = 6) and inflamed (I, n = 8) inflammatory bowel disease (IBD) tissues, determined by TaqMan RT‐qPCR. Statistical analysis: Wilcoxon matched‐pairs signed‐rank test for NI vs. I tissue and control vs. butyrate treatment (B, C); Mann–Whitney test for HC vs. IBD (B). * P < 0.05.

Article Snippet: Tissue and organoid samples were blocked with PBS 1× containing 1% BSA for 30 min at room temperature and incubated overnight at 4 °C with the following primary antibodies: rabbit anti‐human HCAR2 (GPR109A) (1 : 50, Cat No HPA028660, polyclonal), rabbit anti‐human SLC16A1 (MCT1) (1 : 250 for tissue or 1 : 100 for organoids, Cat No HPA003324, polyclonal) (both from Sigma‐Aldrich, Zwijndrecht, The Netherlands), or rabbit anti‐human HDAC3 (1 : 200, Cat No 10255‐1‐AP polyclonal, Proteintech, Rosemont, IL, USA).

Techniques: Expressing, Ex Vivo, Cell Culture, Quantitative RT-PCR, Control, MANN-WHITNEY

Journal: The Febs Journal

Article Title: Butyrate suppresses mucosal inflammation in inflammatory bowel disease primarily through HDAC3 inhibition in monocytes and macrophages

doi: 10.1111/febs.70289

Figure Lengend Snippet: Butyrate induces GPR109A and HDAC3 mRNA expression and reduces IL‐6 and TNF‐α mRNA levels in human inflammatory monocytes in a GPR109A‐independent manner. mRNA levels of HCAR2 and HDAC3 were determined by RT‐qPCR in human peripheral blood monocytes (A, C) and THP‐1 macrophages (B, D) ( n = 4 or n = 6) treated with butyrate in the presence or absence of lipopolysaccharide (LPS) + IFN‐γ (0.1 + 20 ng·mL −1 ) for 6 h. Human peripheral blood monocytes were treated with butyrate (2 m m ) in the presence or absence of LPS + IFN‐γ (0.1 + 20 ng·mL −1 ) for 6 h, with or without pretreatment of 100 μ m mepenzolate bromide (MB) or Pertussis toxin (PT), for 1 h. mRNA levels of (E) IL6 and (F) TNFA ( n = 3 or n = 4) were determined by RT‐qPCR. Statistical analysis: One‐way ANOVA with Bonferroni post‐test (B–D); Wilcoxon matched‐pairs signed‐rank test (A); and paired t ‐test (E, F). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Tissue and organoid samples were blocked with PBS 1× containing 1% BSA for 30 min at room temperature and incubated overnight at 4 °C with the following primary antibodies: rabbit anti‐human HCAR2 (GPR109A) (1 : 50, Cat No HPA028660, polyclonal), rabbit anti‐human SLC16A1 (MCT1) (1 : 250 for tissue or 1 : 100 for organoids, Cat No HPA003324, polyclonal) (both from Sigma‐Aldrich, Zwijndrecht, The Netherlands), or rabbit anti‐human HDAC3 (1 : 200, Cat No 10255‐1‐AP polyclonal, Proteintech, Rosemont, IL, USA).

Techniques: Expressing, Quantitative RT-PCR

Journal: The Febs Journal

Article Title: Butyrate suppresses mucosal inflammation in inflammatory bowel disease primarily through HDAC3 inhibition in monocytes and macrophages

doi: 10.1111/febs.70289

Figure Lengend Snippet: Butyrate induces histone acetylation in THP‐1 macrophages and monocytes and reduces IL‐6 content in human inflammatory monocytes similar as the HDAC3 antagonist SAHA. Acetylation on histone H3 lysine 9 (H3K9ac) was determined intracellularly by flow cytometry in (A) THP‐1 macrophages ( n = 3) and in (B) human peripheral blood monocytes ( n = 5) treated with butyrate or SAHA in the presence or absence of lipopolysaccharide (LPS) + IFN‐γ for 6 h. Secreted IL‐6 (C) and TNF (D) levels by human monocytes ( n = 5) were analyzed using cytometric bead array (CBA). Statistical analysis: Wilcoxon matched‐pairs signed‐rank test and paired t ‐test * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Tissue and organoid samples were blocked with PBS 1× containing 1% BSA for 30 min at room temperature and incubated overnight at 4 °C with the following primary antibodies: rabbit anti‐human HCAR2 (GPR109A) (1 : 50, Cat No HPA028660, polyclonal), rabbit anti‐human SLC16A1 (MCT1) (1 : 250 for tissue or 1 : 100 for organoids, Cat No HPA003324, polyclonal) (both from Sigma‐Aldrich, Zwijndrecht, The Netherlands), or rabbit anti‐human HDAC3 (1 : 200, Cat No 10255‐1‐AP polyclonal, Proteintech, Rosemont, IL, USA).

Techniques: Flow Cytometry