Journal: International Journal of Molecular Sciences

Article Title: Niacin Alleviates Dairy Cow Mastitis by Regulating the GPR109A/AMPK/NRF2 Signaling Pathway

doi: 10.3390/ijms21093321

Figure Lengend Snippet: The relative expression of GPR109A, IL-6, TNF-α , and IL-1β . The mammary glands were collected from healthy dairy cows and mastitis dairy cows ( n = 6). ( a , b ) The results of hematoxylin and eosin (H&E) staining and immunohistochemistry assays in the control and mastitis dairy cows. ( c ) The gene levels of GPR109A in the control and mastitis dairy cows. ( d – f ) The expression of pro-inflammatory factors in the control and mastitis dairy cows. The mRNA levels of GPR109A ( g ), IL-6 ( d ), TNF-α ( e ), and IL-1β ( f ) were normalized to the level of β-actin . The values are presented as the means ± SD (∗ p < 0.05 and ∗∗ p < 0.01).

Article Snippet: AMPK (1:1000, CAT: 5832S), p-AMPK (1:1000, CAT: 2535S), ERK1/2 (1:1000, CAT: 4695S), p-ERK1/2 (1:1000, CAT: 9101L), JNK1/2 (1:1000, CAT: 9252S), p-JNK1/2 (1:1000, CAT: 9251L), P38 (1:1000, CAT: 9212S), p-P38 (1:1000, CAT: 4631S) and NF-κB-p65 (1:1000, CAT: 8242S) were purchased from Cell Signaling Technology (Boston, MA, USA), P62 (WB: 1:1000, Co-IP: 1:50, CAT: 66184-2-2g), LC3B (1:1000, CAT: 18725-1-AP) and Keap1 (1:1000, CAT: 10503-2-AP) were purchased from Proteintech (Rosemont, IL, USA), HO-1(1:1000, CAT: GK284419-11), NRF-2 (WB: 1:1000, IF: 1:150, CAT: Gk298097-1) were purchased from Abcam (Cambridge, UK), GPR109A (WB: 1:200, IHC: 1:50, CAT: NBP1-92180) were purchased from Novus Biologicals (Shanghai, China), β-Tubulin (1:6000, CAT: M05613-4) were purchased from Bosterbio (Pleasanton, CA, USA), IgG (Co-IP: 1 μg, CAT:A7028) were purchased from Beyotime (Shanghai, China), (HRP)-conjugated anti-mouse (1:10,000, CAT: BA1051) or anti-rabbit secondary antibodies (1:10,000, CAT: BA1055) were purchased from Bosterbio (CA, USA).

Techniques: Expressing, Staining, Immunohistochemistry, Control

Journal: International Journal of Molecular Sciences

Article Title: Niacin Alleviates Dairy Cow Mastitis by Regulating the GPR109A/AMPK/NRF2 Signaling Pathway

doi: 10.3390/ijms21093321

Figure Lengend Snippet: Effects of autophagy, GPR109A, and NRF2 on BMECs inflammation. ( a – i ) The bovine mammary epithelial cells (BMECs) were pre-treated with niacin and NC shRNA, shRNA, retinoic acid (RA), or 3-methyladenine (3-MA) for 1 h and then stimulated with LPS for 24 h. The mRNA levels of IL-6, IL-1β , and TNF-α were determined by qRT-PCR. The mRNA levels of IL-6, IL-1β , and TNF-α were normalized to the level of β-actin . The values are presented as the means ± SD (∗ p < 0.05 and ∗∗ p < 0.01).

Article Snippet: AMPK (1:1000, CAT: 5832S), p-AMPK (1:1000, CAT: 2535S), ERK1/2 (1:1000, CAT: 4695S), p-ERK1/2 (1:1000, CAT: 9101L), JNK1/2 (1:1000, CAT: 9252S), p-JNK1/2 (1:1000, CAT: 9251L), P38 (1:1000, CAT: 9212S), p-P38 (1:1000, CAT: 4631S) and NF-κB-p65 (1:1000, CAT: 8242S) were purchased from Cell Signaling Technology (Boston, MA, USA), P62 (WB: 1:1000, Co-IP: 1:50, CAT: 66184-2-2g), LC3B (1:1000, CAT: 18725-1-AP) and Keap1 (1:1000, CAT: 10503-2-AP) were purchased from Proteintech (Rosemont, IL, USA), HO-1(1:1000, CAT: GK284419-11), NRF-2 (WB: 1:1000, IF: 1:150, CAT: Gk298097-1) were purchased from Abcam (Cambridge, UK), GPR109A (WB: 1:200, IHC: 1:50, CAT: NBP1-92180) were purchased from Novus Biologicals (Shanghai, China), β-Tubulin (1:6000, CAT: M05613-4) were purchased from Bosterbio (Pleasanton, CA, USA), IgG (Co-IP: 1 μg, CAT:A7028) were purchased from Beyotime (Shanghai, China), (HRP)-conjugated anti-mouse (1:10,000, CAT: BA1051) or anti-rabbit secondary antibodies (1:10,000, CAT: BA1055) were purchased from Bosterbio (CA, USA).

Techniques: shRNA, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Niacin Alleviates Dairy Cow Mastitis by Regulating the GPR109A/AMPK/NRF2 Signaling Pathway

doi: 10.3390/ijms21093321

Figure Lengend Snippet: Niacin can activate the GPR109A/NRF2/autophagy signal pathway. The cells were collected at 0, 3, 6, 12, and 24 h to extract the total protein. The total protein was prepared and subjected to Western blotting using GPR109A, NRF-2, HO-1, and β-tubulin antibodies. T-NRF-2 means total NRF-2. ( a – d ) The protein levels of GPR109A, NRF-2, and HO-1. The cells from different experimental groups were treated with niacin or shRNA+niacin for 24 h, and then, the total protein was collected. N-NRF-2 means NRF-2 in the nucleus. C-NRF-2 means NRF-2 in the cytoplasm. ( e – h ) The protein levels of GPR109A, C-NRF-2, N-NRF-2, and HO-1. Each immunoreactive band was digitized and expressed as a ratio of the β-tubulin level. ( i ) The immunofluorescence results of the assay for NRF-2. The scale length in the figure is 200 μM. ( j ) The relative fluorescence intensity of NRF-2/ARE. The mRNA levels were determined by qRT-PCR. ( k ) The mRNA levels of ATG12, ATG4D, p62, ATG5, ULK1, ATG4B, Beclin, LC3B , and ATG7 were normalized to the level of β-actin . The values are presented as the means ± SD (∗ p < 0.05 and ∗∗ p < 0.01).

Article Snippet: AMPK (1:1000, CAT: 5832S), p-AMPK (1:1000, CAT: 2535S), ERK1/2 (1:1000, CAT: 4695S), p-ERK1/2 (1:1000, CAT: 9101L), JNK1/2 (1:1000, CAT: 9252S), p-JNK1/2 (1:1000, CAT: 9251L), P38 (1:1000, CAT: 9212S), p-P38 (1:1000, CAT: 4631S) and NF-κB-p65 (1:1000, CAT: 8242S) were purchased from Cell Signaling Technology (Boston, MA, USA), P62 (WB: 1:1000, Co-IP: 1:50, CAT: 66184-2-2g), LC3B (1:1000, CAT: 18725-1-AP) and Keap1 (1:1000, CAT: 10503-2-AP) were purchased from Proteintech (Rosemont, IL, USA), HO-1(1:1000, CAT: GK284419-11), NRF-2 (WB: 1:1000, IF: 1:150, CAT: Gk298097-1) were purchased from Abcam (Cambridge, UK), GPR109A (WB: 1:200, IHC: 1:50, CAT: NBP1-92180) were purchased from Novus Biologicals (Shanghai, China), β-Tubulin (1:6000, CAT: M05613-4) were purchased from Bosterbio (Pleasanton, CA, USA), IgG (Co-IP: 1 μg, CAT:A7028) were purchased from Beyotime (Shanghai, China), (HRP)-conjugated anti-mouse (1:10,000, CAT: BA1051) or anti-rabbit secondary antibodies (1:10,000, CAT: BA1055) were purchased from Bosterbio (CA, USA).

Techniques: Western Blot, shRNA, Immunofluorescence, Fluorescence, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Niacin Alleviates Dairy Cow Mastitis by Regulating the GPR109A/AMPK/NRF2 Signaling Pathway

doi: 10.3390/ijms21093321

Figure Lengend Snippet: GPR109A regulates energy metabolism, AMPK phosphorylation, and P62 interact with Keap-1 in BMECs. The cells were collected at 0, 3, 6, 12, and 24 h to extract the total protein. ( a , b ) The protein levels of p-AMPK. The cells from different experimental groups were treated with niacin or shRNA + niacin for 24 h, and then, the total protein was collected. The cell lysates were prepared and subjected to Western blotting using AMPK and p-AMPK antibodies. ( c , d ) The protein levels of p-AMPK. Each immunoreactive band was digitized and expressed as a ratio of the β-tubulin. ( e ) The BMECs were pre-treated with niacin, niacin+shRNA, or shRNA for 24 h. The cells were then collected, and the ATP, ADP, and AMP levels were detected by liquid chromatography ( n = 3). ( f – h ) of the ATP, ADP, and AMP content in the BMECs after adding niacin, niacin + shRNA or shRNA. ( i ) The ratio of (AMP + ADP)/ATP. The values are presented as the means ± SD (ns means no difference, ∗ p < 0.05 and ∗∗ p < 0.01).

Article Snippet: AMPK (1:1000, CAT: 5832S), p-AMPK (1:1000, CAT: 2535S), ERK1/2 (1:1000, CAT: 4695S), p-ERK1/2 (1:1000, CAT: 9101L), JNK1/2 (1:1000, CAT: 9252S), p-JNK1/2 (1:1000, CAT: 9251L), P38 (1:1000, CAT: 9212S), p-P38 (1:1000, CAT: 4631S) and NF-κB-p65 (1:1000, CAT: 8242S) were purchased from Cell Signaling Technology (Boston, MA, USA), P62 (WB: 1:1000, Co-IP: 1:50, CAT: 66184-2-2g), LC3B (1:1000, CAT: 18725-1-AP) and Keap1 (1:1000, CAT: 10503-2-AP) were purchased from Proteintech (Rosemont, IL, USA), HO-1(1:1000, CAT: GK284419-11), NRF-2 (WB: 1:1000, IF: 1:150, CAT: Gk298097-1) were purchased from Abcam (Cambridge, UK), GPR109A (WB: 1:200, IHC: 1:50, CAT: NBP1-92180) were purchased from Novus Biologicals (Shanghai, China), β-Tubulin (1:6000, CAT: M05613-4) were purchased from Bosterbio (Pleasanton, CA, USA), IgG (Co-IP: 1 μg, CAT:A7028) were purchased from Beyotime (Shanghai, China), (HRP)-conjugated anti-mouse (1:10,000, CAT: BA1051) or anti-rabbit secondary antibodies (1:10,000, CAT: BA1055) were purchased from Bosterbio (CA, USA).

Techniques: Phospho-proteomics, shRNA, Western Blot, Liquid Chromatography

Journal: International Journal of Molecular Sciences

Article Title: Niacin Alleviates Dairy Cow Mastitis by Regulating the GPR109A/AMPK/NRF2 Signaling Pathway

doi: 10.3390/ijms21093321

Figure Lengend Snippet: GPR109A alleviates inflammation of BMECs by regulating autophagy. Cells from different experimental groups were treated with niacin, LPS, or niacin+LPS for 24 h, and then, the total protein was collected. The cell lysates were prepared and subjected to Western blotting using β-tubulin ( a ), LC3B ( a , b ), and P62 ( a , c ) antibodies. Each immunoreactive band was digitized and expressed as a ratio of the β-tubulin. ( d ) The left panel shows 6000 × 120 V; the right panel shows is 12,000 × 120 V. The results revealed by the electron microscopy showed that niacin could induce autophagy in the non-treated BMECs and LPS-treated BMECs. ( e – m ) The mRNA levels were determined by qRT-PCR. The mRNA levels of ATG12, ATG4D , p62 , ATG5, ULK1, ATG4B, Beclin, LC3B , and ATG7 were normalized to the level of β-actin . Values are presented as the means ± SD (∗ p < 0.05 and ∗∗ p < 0.001).

Article Snippet: AMPK (1:1000, CAT: 5832S), p-AMPK (1:1000, CAT: 2535S), ERK1/2 (1:1000, CAT: 4695S), p-ERK1/2 (1:1000, CAT: 9101L), JNK1/2 (1:1000, CAT: 9252S), p-JNK1/2 (1:1000, CAT: 9251L), P38 (1:1000, CAT: 9212S), p-P38 (1:1000, CAT: 4631S) and NF-κB-p65 (1:1000, CAT: 8242S) were purchased from Cell Signaling Technology (Boston, MA, USA), P62 (WB: 1:1000, Co-IP: 1:50, CAT: 66184-2-2g), LC3B (1:1000, CAT: 18725-1-AP) and Keap1 (1:1000, CAT: 10503-2-AP) were purchased from Proteintech (Rosemont, IL, USA), HO-1(1:1000, CAT: GK284419-11), NRF-2 (WB: 1:1000, IF: 1:150, CAT: Gk298097-1) were purchased from Abcam (Cambridge, UK), GPR109A (WB: 1:200, IHC: 1:50, CAT: NBP1-92180) were purchased from Novus Biologicals (Shanghai, China), β-Tubulin (1:6000, CAT: M05613-4) were purchased from Bosterbio (Pleasanton, CA, USA), IgG (Co-IP: 1 μg, CAT:A7028) were purchased from Beyotime (Shanghai, China), (HRP)-conjugated anti-mouse (1:10,000, CAT: BA1051) or anti-rabbit secondary antibodies (1:10,000, CAT: BA1055) were purchased from Bosterbio (CA, USA).

Techniques: Western Blot, Electron Microscopy, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Niacin Alleviates Dairy Cow Mastitis by Regulating the GPR109A/AMPK/NRF2 Signaling Pathway

doi: 10.3390/ijms21093321

Figure Lengend Snippet: Effect of GPR109A on the AMPK/NRF-2/HO-1, P38, JNK1/2, and ERK1/2 signaling pathways. The cells from different experimental groups were treated with niacin, niacin, or LPS for 24 h, and then, the total protein was collected. N-P65 indicates P65 in the nucleus. N-NRF-2 means NRF-2 in the nucleus. The cell lysates were prepared and subjected to Western blotting using antibodies for P38 ( a , d ), p-P38 ( a , d ), JNK1/2 ( a , b ), p-JNK1/2 ( a , b ), ERK1/2 ( a , c ), p-ERK1/2 ( a , c ), AMPK ( a , f ), p-AMPK ( a , f ), HO-1 ( a , g ), β-tubulin ( e ), N-P65 ( a , h ), N-NRF-2 ( a , i – k ), and laminin B ( e ).

Article Snippet: AMPK (1:1000, CAT: 5832S), p-AMPK (1:1000, CAT: 2535S), ERK1/2 (1:1000, CAT: 4695S), p-ERK1/2 (1:1000, CAT: 9101L), JNK1/2 (1:1000, CAT: 9252S), p-JNK1/2 (1:1000, CAT: 9251L), P38 (1:1000, CAT: 9212S), p-P38 (1:1000, CAT: 4631S) and NF-κB-p65 (1:1000, CAT: 8242S) were purchased from Cell Signaling Technology (Boston, MA, USA), P62 (WB: 1:1000, Co-IP: 1:50, CAT: 66184-2-2g), LC3B (1:1000, CAT: 18725-1-AP) and Keap1 (1:1000, CAT: 10503-2-AP) were purchased from Proteintech (Rosemont, IL, USA), HO-1(1:1000, CAT: GK284419-11), NRF-2 (WB: 1:1000, IF: 1:150, CAT: Gk298097-1) were purchased from Abcam (Cambridge, UK), GPR109A (WB: 1:200, IHC: 1:50, CAT: NBP1-92180) were purchased from Novus Biologicals (Shanghai, China), β-Tubulin (1:6000, CAT: M05613-4) were purchased from Bosterbio (Pleasanton, CA, USA), IgG (Co-IP: 1 μg, CAT:A7028) were purchased from Beyotime (Shanghai, China), (HRP)-conjugated anti-mouse (1:10,000, CAT: BA1051) or anti-rabbit secondary antibodies (1:10,000, CAT: BA1055) were purchased from Bosterbio (CA, USA).

Techniques: Protein-Protein interactions, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Niacin Alleviates Dairy Cow Mastitis by Regulating the GPR109A/AMPK/NRF2 Signaling Pathway

doi: 10.3390/ijms21093321

Figure Lengend Snippet: The primer sequenceof the genes.

Article Snippet: AMPK (1:1000, CAT: 5832S), p-AMPK (1:1000, CAT: 2535S), ERK1/2 (1:1000, CAT: 4695S), p-ERK1/2 (1:1000, CAT: 9101L), JNK1/2 (1:1000, CAT: 9252S), p-JNK1/2 (1:1000, CAT: 9251L), P38 (1:1000, CAT: 9212S), p-P38 (1:1000, CAT: 4631S) and NF-κB-p65 (1:1000, CAT: 8242S) were purchased from Cell Signaling Technology (Boston, MA, USA), P62 (WB: 1:1000, Co-IP: 1:50, CAT: 66184-2-2g), LC3B (1:1000, CAT: 18725-1-AP) and Keap1 (1:1000, CAT: 10503-2-AP) were purchased from Proteintech (Rosemont, IL, USA), HO-1(1:1000, CAT: GK284419-11), NRF-2 (WB: 1:1000, IF: 1:150, CAT: Gk298097-1) were purchased from Abcam (Cambridge, UK), GPR109A (WB: 1:200, IHC: 1:50, CAT: NBP1-92180) were purchased from Novus Biologicals (Shanghai, China), β-Tubulin (1:6000, CAT: M05613-4) were purchased from Bosterbio (Pleasanton, CA, USA), IgG (Co-IP: 1 μg, CAT:A7028) were purchased from Beyotime (Shanghai, China), (HRP)-conjugated anti-mouse (1:10,000, CAT: BA1051) or anti-rabbit secondary antibodies (1:10,000, CAT: BA1055) were purchased from Bosterbio (CA, USA).

Techniques: Sequencing

Journal: British journal of pharmacology

Article Title: 4-Hydroxynonenal-induced GPR109A (HCA 2 receptor) activation elicits bipolar responses, G αi -mediated anti-inflammatory effects and G βγ -mediated cell death.

doi: 10.1111/bph.14174

Figure Lengend Snippet: Figure 1 GPR109A activation by 4-HNE, niacin and 3-OHBA induces apoptotic death in ARPE-19 and CCD-841 cells. (A) GPR109A and NOX4 expression levels were analysed by immunoblotting. Bar graphs represent averaged quantitations of immunoblots from six independent experiments. (B–F) Cell viability was measured via MTT assay. (G) ARPE-19 cells were stained with Hoechst 33258, a water-soluble DNA-binding compound, and 6-carboxyfluroscein diacetate, a secondary dye that accumulates in viable cells. (H) Annexin V-positive and PI-positive ARPE-19 cell popula- tions were determined via flow cytometry. (I) Caspase-3/7 activity was measured in 4-HNE-treated ARPE-19 cells using a Caspase-Glo® 3/7 assay kit. *P < 0.05 versus vehicle-treated controls.

Article Snippet: Monoclonal antibody against human GPR109A was obtained from Novus Biological (Cat. No. NBP2-12358AF488; Littleton, CO, USA).

Techniques: Activation Assay, Expressing, Western Blot, MTT Assay, Staining, Binding Assay, Cytometry, Activity Assay, Caspase-Glo Assay

Journal: British journal of pharmacology

Article Title: 4-Hydroxynonenal-induced GPR109A (HCA 2 receptor) activation elicits bipolar responses, G αi -mediated anti-inflammatory effects and G βγ -mediated cell death.

doi: 10.1111/bph.14174

Figure Lengend Snippet: Figure 7 4-HNE is a stronger agonist than niacin, not competing with niacin for binding sites in GPR109A. (A) A functional activity assay using the CHO-K1- GPR109A-Gi cell line, which was designed to detect the inhibition of cAMP generation, was performed in the cells pre-stimulated with forskolin prior to treatment with the other agonists. (B) 4-HNE, niacin, 3-OHBA did not induce cytotoxicity in CHO-K1-GPR109A-Gi cells. (C) SPR binding assay showing 4-HNE and niacin bind to GPR109A in a concentration-dependent manner. (D) [3H]-niacin binding competition assay with 4-HNE and niacin showing their effects are concentration-dependent in HEK-293T cells stably expressing human GPR109A. (E and F) CHO-K1 cells were transfected with five different FLAG-tagged plasmids of GPR109A mutants (R111A, R253A, W256A, F277A and L280A). (E) Indicates their trans- fection efficiency as demonstrated by FLAG staining and (F) shows the results of the functional activity assay indicating that 4-HNE and niacin de- creased the forskolin-induced production of cAMP in a concentration-dependent manner. (G–I) Effects of combination treatments with the GPR109A ligands (niacin, 3-OHBA or 4-HNE). (G) CHO-K1-GPR109A cells were treated with a combination of two from three ligands, and forskolin-induced cAMP levels were measured. In ARPE-19 cells, effects of combination treatments on changes in (H) intracellular Ca2+ levels and (I) cell viability were determined. *P < 0.05 versus vehicle-treated controls. #P < 0.05 versus niacin-treated cells. $P < 0.05 versus 3-OHBA- treated cells. &P < 0.05 versus 4-HNE-treated cells. RU, response units.

Article Snippet: Monoclonal antibody against human GPR109A was obtained from Novus Biological (Cat. No. NBP2-12358AF488; Littleton, CO, USA).

Techniques: Binding Assay, Functional Assay, Activity Assay, Inhibition, Concentration Assay, Competitive Binding Assay, Stable Transfection, Expressing, Transfection, Staining

Journal: Cell death and differentiation

Article Title: Neutrophil apoptosis mediated by nicotinic acid receptors (GPR109A).

doi: 10.1038/sj.cdd.4402238

Figure Lengend Snippet: Figure 1 Mature blood neutrophils, but not immature bone marrow neutrophils or mature blood eosinophils, express the NA receptor GPR109A on their surface. (a) Flow cytometry. Cells were stained with control (black) or anti-GPR109A (green) mAbs. (b and c) Confocal microscopy. Neutrophils were stained with FITC- conjugated NA and anti-GPR109A mAb, respectively. Nuclei were stained with PE in (b). Results in each panel are representative of at least four independent experiments

Article Snippet: Anti-GPR109A (HM74a) rat mAb was from R&D Systems Europe Ltd. (Abingdon, UK).

Techniques: Flow Cytometry, Staining, Control, Confocal Microscopy