Journal: Drug Design, Development and Therapy

Article Title: Modified Zuo Gui Wan Ameliorates Ovariectomy-Induced Osteoporosis in Rats by Regulating the SCFA-GPR41-p38MAPK Signaling Pathway

doi: 10.2147/DDDT.S482965

Figure Lengend Snippet: Effect of MZGW on serum SCFA level and the relative expression of tibia CTSK, NFATc1, GPR41, and p-p38 proteins in OVX rats. ( A ) Western blot image of NFATc1 and CTSK protein expression. ( B ) Relative protein expression of NFATc1. ( C ) Relative protein expression of CTSK. ( D ) Changes in the total serum SCFA levels per group. ( E ) Western blot image of GPR41 protein expression. ( F ) Relative protein expression of GPR41. ( G ) Western blot image of p-p38 and p38 protein expression. ( H ) Relative protein expression of p-p38. ( I ) Relative mRNA expression of p38 examined using quantitative reverse transcription PCR (qRT-PCR). Data were expressed as mean ± SD, n=6. # p <0.05 and ## p <0.01 vs the SHAM group; * p <0.05 and ** p <0.01 vs the OVX group.

Article Snippet: After electrophoresis with 8% SDS-PAGE (APPLYGEN), proteins were transferred to a PVDF membrane and blocked with 5% skimmed milk/BSA (APPLYGEN) for 1 h. Subsequently, proteins were incubated with primary antibodies against β-actin (1:10,000, Proteintech, China), GPR41 (1:500, Proteintech), p38 (1:1000, Proteintech), p-p38 (1:1000, Proteintech), CTSK (1:1000, Proteintech), and NFATc1 (1:1000, Proteintech) at 4°C overnight, followed by washing three times rapidly with TBST.

Techniques: Expressing, Western Blot, Reverse Transcription, Quantitative RT-PCR

Journal: Drug Design, Development and Therapy

Article Title: Modified Zuo Gui Wan Ameliorates Ovariectomy-Induced Osteoporosis in Rats by Regulating the SCFA-GPR41-p38MAPK Signaling Pathway

doi: 10.2147/DDDT.S482965

Figure Lengend Snippet: SCFAs inhibited osteoclast differentiation and decrease the expression of mRNA and proteins associated with the SCFA-GPR41-p38MAPK signaling pathway. ( A ) Representative images of TRAP-stained osteoclasts depicting osteoblast differentiation in each group (scale bar=100 μm). ( B ) Quantitative statistics of osteoclast number (n=6). ( C ) Representative images of toluidine blue-stained osteoclasts in each group depicting bone resorption function. Differentiation of osteoblasts in each group (scale bar=100 μm). ( D ) Measurement of the area of bone resorption lacunae using Image-pro-plus 6.0 software. Data are expressed as mean ± SD, n=6. ( E ) Western blot image of GPR41, p-p38, CTSK, and NFATc1 protein expression. ( F ) Relative protein expression of GPR41. ( G ) Relative protein expression of p-p38. ( H ) Relative mRNA expression of p38. ( I ) Relative protein expression of CTSK. ( J ) Relative protein expression of NFATc1. Data are expressed as mean ± SD, n=6. # p <0.05 and ## p <0.01 vs the NC-S group; ** p <0.01 vs the OVX-S group; ◇ p <0.05 and ◇◇ p <0.01 vs the MZGW-DS group; ☆☆ p <0.01 vs the NC- SCFAs group; Δ p <0.05 and ΔΔ p <0.01 vs the OVX-SCFAs group.

Article Snippet: After electrophoresis with 8% SDS-PAGE (APPLYGEN), proteins were transferred to a PVDF membrane and blocked with 5% skimmed milk/BSA (APPLYGEN) for 1 h. Subsequently, proteins were incubated with primary antibodies against β-actin (1:10,000, Proteintech, China), GPR41 (1:500, Proteintech), p38 (1:1000, Proteintech), p-p38 (1:1000, Proteintech), CTSK (1:1000, Proteintech), and NFATc1 (1:1000, Proteintech) at 4°C overnight, followed by washing three times rapidly with TBST.

Techniques: Expressing, Staining, Software, Western Blot

Journal: Drug Design, Development and Therapy

Article Title: Modified Zuo Gui Wan Ameliorates Ovariectomy-Induced Osteoporosis in Rats by Regulating the SCFA-GPR41-p38MAPK Signaling Pathway

doi: 10.2147/DDDT.S482965

Figure Lengend Snippet: Knockdown of the GPR41 gene, a key receptor for SCFAs binding in the SCFA-GPR41-p38MAPK pathway, did not reverse osteoclast-induced bone destruction by MZGW drug-containing serum (MZGW-DS). ( A ) Transfection efficiency of knocked down GPR41 protein detected using Western blotting. ( B ) Relative protein expression of GPR41. Data are expressed as mean ± SD, n=6. * p <0.05 and ** p <0.01 vs the OC+siNC group. ( C ) Western blot image depicting GPR41 protein expression upon knockdown of the GPR41 gene. ( D ) Relative protein expression of GPR41. ( E ) Relative expression of p38mRNA in osteoclasts in each group. ( F ) Western blot image depicting p-p38 and p38 protein expression upon knockdown of the GPR41 gene. ( G ) Relative protein expression of p-p38 and p38. ( H ) Quantitative statistics of osteoclast number (n=6). ( I ) Representative images of TRAP-stained osteoclasts in each group (scale bar=100 μm) upon knockdown of the GPR41 gene. ( J ) Western blot image of CTSK protein. ( K ) Relative protein expression of CTSK. ( L ) Western blot image of NFATc1 protein. ( M ) Relative protein expression of NFATc1. Data are expressed as mean ± SD, n=6. # p <0.05 and ## p <0.01 vs the OC+siNC group; * p <0.05 and ** p <0.01 vs the OC+siGPR41 group; ΔΔ p <0.01 vs the OC+siNC+MZGW-DS group.

Article Snippet: After electrophoresis with 8% SDS-PAGE (APPLYGEN), proteins were transferred to a PVDF membrane and blocked with 5% skimmed milk/BSA (APPLYGEN) for 1 h. Subsequently, proteins were incubated with primary antibodies against β-actin (1:10,000, Proteintech, China), GPR41 (1:500, Proteintech), p38 (1:1000, Proteintech), p-p38 (1:1000, Proteintech), CTSK (1:1000, Proteintech), and NFATc1 (1:1000, Proteintech) at 4°C overnight, followed by washing three times rapidly with TBST.

Techniques: Knockdown, Binding Assay, Transfection, Western Blot, Expressing, Staining

Journal: eLife

Article Title: Short-chain fatty acids activate acetyltransferase p300

doi: 10.7554/eLife.72171

Figure Lengend Snippet:

Article Snippet: antibody , FFAR3/GPR41, rabbit , Novus Biologicals , NBP2-14014 , 1:1,000 dilution.

Techniques: Mutagenesis, Transfection, Construct

Journal: Scientific Reports

Article Title: Human and mouse monocytes display distinct signalling and cytokine profiles upon stimulation with FFAR2/FFAR3 short-chain fatty acid receptor agonists

doi: 10.1038/srep34145

Figure Lengend Snippet: Human tissue sections were probed via immunohistochemistry. The merged color (blue), is the result of the colocalization of antibody staining for FFAR2 or FFAR3 (both stained green) with the CD163 monocytes/macrophage marker (stained red). ( a ) Colon epithelial cells, liver hepatocytes, and the CD163+ spleen monocytes/macrophages, stained positive for FFAR2 and FFA3. Only weak FFAR2 and FFAR3 staining was observed for the CD163+ monocytes/macrophages of the colon, liver, and lung. ( b ) CD163+ peripheral blood monocytes stained positive for FFAR2 and FFAR3, and this staining was reduced when a portion of the monocyte sample was differentiated in vitro into macrophages. The data shown are representative of three independent experiments.

Article Snippet: Following antigen retrieval (0.01 M Citrate buffer pH 6, 20 min at 99 °C), tissue sections were stained at 4 °C overnight with primary antibodies against CD163 (Thermo Scientific, MA5-11458), FFAR3 (Atlas Antibodies, HPA044681, Lot R40941) and FFAR2 (Santa Cruz Biotechnology, sc-32906).

Techniques: Immunohistochemistry, Staining, Marker, In Vitro

Journal: Scientific Reports

Article Title: Human and mouse monocytes display distinct signalling and cytokine profiles upon stimulation with FFAR2/FFAR3 short-chain fatty acid receptor agonists

doi: 10.1038/srep34145

Figure Lengend Snippet: Human monocytes were treated with either 5 mM acetate or 10 μM CFMB (FFAR2 agonist) or 10 μM AR420626 (FFAR3 agonist) or the respective solvent controls (NT, 0.1% v/v DMSO) for 15 min followed by 100 ng/mL LPS challenge for 6 h and 12 h. ( a ) Cytokine proteome arrays detect reduced C5, CCL1, CCL2, GM-CSF, IL-1α, IL-1β and ICAM-1 expression in acetate treated monocytes. Monocytes treated with either CFMB or AR420626 displayed reduced C5, CCL2 and IL-1β expression. Two independent cultures for each treatment condition is shown; n = 2. ( b ) Acetate- and synthetic agonist- mediated inhibition of IL-1β and CCL2 was confirmed by Enzyme-linked immunosorbent assay (ELISA). The data shown are the mean concentration from three independent cultures for each treatment condition (±SEM; n = 3) and are representative of three independent experiments.

Article Snippet: Following antigen retrieval (0.01 M Citrate buffer pH 6, 20 min at 99 °C), tissue sections were stained at 4 °C overnight with primary antibodies against CD163 (Thermo Scientific, MA5-11458), FFAR3 (Atlas Antibodies, HPA044681, Lot R40941) and FFAR2 (Santa Cruz Biotechnology, sc-32906).

Techniques: Solvent, Expressing, Inhibition, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Scientific Reports

Article Title: Human and mouse monocytes display distinct signalling and cytokine profiles upon stimulation with FFAR2/FFAR3 short-chain fatty acid receptor agonists

doi: 10.1038/srep34145

Figure Lengend Snippet: ( a ) Human monocytes upregulated inflammatory cytokines upon LPS challenge. ( b ) Human monocytes display reduced CCL1 , CCL2 , CCL3 , GM-CSF , IL1 α, IL1 β and ICAM1 upon acetate or FFAR2 and FFAR3 synthetic agonist treatment. ( c ) Acetate- and FFAR2 synthetic agonist-induced cytokine repression is observed in both naive and activated monocytes after 4 h of induction. ( a–c ) Unless otherwise indicated, human monocytes were treated with either 5 mM acetate, 10 μM CFMB (FFAR2 agonist), 10 μM AR420626 (FFAR3 agonist) or the respective solvent control for 15 min followed by a 4 h activation with inflammatory stimuli before the cytokine mRNA levels were measured by real-time analysis. Inflammatory stimuli: 100 ng/mL LPS, 10 ng/mL TNF, 200 nM 12-O-Tetradecanoylphorbol-13-acetate (TPA) and 100 ng/mL PAM3CSK4. The data shown are the means of three independent cultures for each treatment condition and is presented as the fold change of the acetate/CFMB/AR420626 treated samples relative to the respective solvent controls ± SEM; n = 3. The data shown are representative of three independent experiments. n.d: non-detectable. The two tailed Welch’s t-test was used to determine the statistical significance of the fold change (between the agonist treatment group and controls) and is annotated as: *<0.05, **<0.005, and ***<0.0005.

Article Snippet: Following antigen retrieval (0.01 M Citrate buffer pH 6, 20 min at 99 °C), tissue sections were stained at 4 °C overnight with primary antibodies against CD163 (Thermo Scientific, MA5-11458), FFAR3 (Atlas Antibodies, HPA044681, Lot R40941) and FFAR2 (Santa Cruz Biotechnology, sc-32906).

Techniques: Solvent, Control, Activation Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: Human and mouse monocytes display distinct signalling and cytokine profiles upon stimulation with FFAR2/FFAR3 short-chain fatty acid receptor agonists

doi: 10.1038/srep34145

Figure Lengend Snippet: ( a , b ) Mouse monocytes were treated with either 5 mM acetate or the solvent control for 15 min followed by 100 ng/mL LPS challenge for 12 h. ( a ) Cytokine expression analysis via proteome arrays. Following acetate treatment, mouse peripheral blood monocytes displayed reduced CXCL10, IL-17 and TMIP-1 expression and elevated CCL1, CCL3, GM-CSF, IL-1α, IL-1β and TNF, when compared to control monocytes treated with LPS alone. Wild type (WT), Ffar2 −/− , and Ffar3 −/− mouse bone marrow monocytes, responded to acetate with reduced CXCL10, IL-1rα, TIMP-1 and CCL2, and elevated CXCL1, IL-1α and IL-1β. Two independent cultures for each treatment condition is shown; n = 2. ( b ) Real-time PCR analysis showing changes in cytokine mRNA in mouse bone marrow monocytes (WT, Ffar2 −/− and Ffar3 −/− ) treated with acetate. The data shown are the means of three independent cultures for each treatment condition and is presented as the fold change of the acetate treated samples relative to the solvent controls ± SEM; n = 3. n.d: non-detectable. The data shown are representative of three independent experiments. ( c ) P38 phosphorylation in mouse bone marrow monocytes during 5 mM acetate treatment. Images are cropped for clarity; full-length blots are presented in . ( d ) Western blot of mouse bone marrow monocytes treated with 5 mM acetate for 15 min followed by 100 ng/mL LPS challenge. No change in Akt and ERK phosphorylation levels was detected compared to controls treated with LPS alone. Images are cropped for clarity; full-length blots are presented in .

Article Snippet: Following antigen retrieval (0.01 M Citrate buffer pH 6, 20 min at 99 °C), tissue sections were stained at 4 °C overnight with primary antibodies against CD163 (Thermo Scientific, MA5-11458), FFAR3 (Atlas Antibodies, HPA044681, Lot R40941) and FFAR2 (Santa Cruz Biotechnology, sc-32906).

Techniques: Solvent, Control, Expressing, Real-time Polymerase Chain Reaction, Phospho-proteomics, Western Blot

Journal: Scientific Reports

Article Title: Human and mouse monocytes display distinct signalling and cytokine profiles upon stimulation with FFAR2/FFAR3 short-chain fatty acid receptor agonists

doi: 10.1038/srep34145

Figure Lengend Snippet: ( a ) Western blot shows that heterologous expression of FFAR2 leads to the constitutive phosphorylation of p38. A549 cells were transfected with FFAR2-pcDNA or FFAR3-pcDNA or the pcDNA control vector. Images are cropped for clarity; full-length blots are presented in . ( b ) The induction of NF-κB by TNF is reduced in A549 cells that overexpress FFAR2 versus the control cells transfected with the pcDNA control vector. ( c ) Inhibition of NF-κB activity is proportional to FFAR2 expression levels as measured by NF-κB luciferase assay. ( d ) The inhibition of NF-κB during FFAR2 expression is not affected by BisI, a PKC inhibitor. A549 cells were treated with BisI during transfection for 24 h. ( e ) The inhibition of NF-κB during FFAR2 expression is diminished when a constitutively active form of Akt (myrAkt) is present. ( f ) Among human and mouse FFAR2 and FFAR3, overexpression of human FFAR2 in A549 resulted in the strongest inhibition of NF-κB activation by TNF. ( b–f ) NF-κB luciferase reporter assays. Cells were transfected with either the FFAR2/3-pcDNA or the pcDNA control vector together with the NF-κB dual luciferase reporter plasmids. 24 h after transfection, cells were treated with 5 ng/mL TNF for 6 h before the luciferase reporters were assayed. NF-κB activity values are expressed as relative fold changes to the control cells (pcDNA control vector, no treatment). Values shown are the average of three independent cultures (n = 3) with the error bars representing mean ± SEM. The two tailed Welch’s t-test was used to determine statistical significance and is annotated as *<0.05. The data shown are representative of three independent experiments.

Article Snippet: Following antigen retrieval (0.01 M Citrate buffer pH 6, 20 min at 99 °C), tissue sections were stained at 4 °C overnight with primary antibodies against CD163 (Thermo Scientific, MA5-11458), FFAR3 (Atlas Antibodies, HPA044681, Lot R40941) and FFAR2 (Santa Cruz Biotechnology, sc-32906).

Techniques: Western Blot, Expressing, Phospho-proteomics, Transfection, Control, Plasmid Preparation, Inhibition, Activity Assay, Luciferase, Over Expression, Activation Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: Human and mouse monocytes display distinct signalling and cytokine profiles upon stimulation with FFAR2/FFAR3 short-chain fatty acid receptor agonists

doi: 10.1038/srep34145

Figure Lengend Snippet: The related SCFA receptors, FFAR2 and FFAR3, were found to mediate human monocyte response to SCFAs by suppressing inflammatory signalling and the resulting cytokine expression. Surprisingly, FFAR2 and FFAR3 signalling is divergent in mouse monocytes, resulting in response to acetate treatment that is distinct from human monocytes.

Article Snippet: Following antigen retrieval (0.01 M Citrate buffer pH 6, 20 min at 99 °C), tissue sections were stained at 4 °C overnight with primary antibodies against CD163 (Thermo Scientific, MA5-11458), FFAR3 (Atlas Antibodies, HPA044681, Lot R40941) and FFAR2 (Santa Cruz Biotechnology, sc-32906).

Techniques: Expressing

Journal: Scientific Reports

Article Title: Human and mouse monocytes display distinct signalling and cytokine profiles upon stimulation with FFAR2/FFAR3 short-chain fatty acid receptor agonists

doi: 10.1038/srep34145

Figure Lengend Snippet: List of PCR primers.

Article Snippet: Following antigen retrieval (0.01 M Citrate buffer pH 6, 20 min at 99 °C), tissue sections were stained at 4 °C overnight with primary antibodies against CD163 (Thermo Scientific, MA5-11458), FFAR3 (Atlas Antibodies, HPA044681, Lot R40941) and FFAR2 (Santa Cruz Biotechnology, sc-32906).

Techniques:

Journal: Frontiers in Endocrinology

Article Title: The short-chain fatty acid receptors Gpr41/43 regulate bone mass by promoting adipogenic differentiation of mesenchymal stem cells

doi: 10.3389/fendo.2024.1392418

Figure Lengend Snippet: Expression analysis of Gpr41 and Gpr43 and body composition of Gpr41/43 dKO mice at different time points of age. (A) qRT-PCR expression analysis of Gpr41 and (B) Gpr43 in indicated tissues and primary osteoblasts, osteoclasts and chondrocytes at different differentiation time points relative to the respective Gapdh expression. n= 3 samples per tissue from 10 weeks old female C57Bl/6J. WT (white bars) and Gpr41/43 dKO (blue bars) mice were used to investigate body composition and organ weights at 6 (n= 5 WT, n=5 Grp41/43 dKO), 13 (n=7 WT, n=5 Gpr41/43 dKO) and 30 (n=3 WT, n=4-5 Gpr41/43 dKO) weeks of age. (C) Study design: WT and Gpr41/43 dKO mice were investigated at 6, 13 and 30 weeks of age and read outs were (D) body weight, (E) fat mass rel. to body weight (BW), (F) lean mass rel. to BW, (G) liver weight, (H) inguinal white adipose tissue (ingWAT), (I) epididymal white adipose tissue (epiWAT). (J) At indicated time points after gavage, glucose concentrations and (K) glucose quantification (area under the curve (AUC)) were determined in blood from tail vein of WT (n=4) and Gpr41/43 dKO (n=4) mice. Data were shown as dot plots with median values indicated as horizontal bars ± SEM analyzed by Student’s t-test. *p < 0.05 vs. Gpr41/43 dKO, **p < 0.01 vs. Gpr41/43 dKO.

Article Snippet: Quantitative real-time PCR was performed with a QuantStudioTM 5 Real-Time PCR System (Thermo Fisher Scientific) using assays-on-demand primer (Applied Biosystems, assay IDs: Acp5: Mm00475698_m1, Clcn7: Mm00442400_m1, Ctsk: Mm00484039_m1, Col1a1 : Mm00801666_g1, Adipoq : Mm00456425_m1, Eno3 : Mm00468267_m1, Gpr41 : Mm02621638_s1, Gpr43 : Mm02620654_s1, Kcnk1 : Mm00492791_m1, Pparg : Mm00440945_m1, Phex : Mm00448119_m1, Ptgs2 : Mm00478374_m1, Lpl : Mm00434764_m1) that were premixed with TaqMan Universal MasterMix II (Applied Biosystems).

Techniques: Expressing, Quantitative RT-PCR

Journal: Frontiers in Endocrinology

Article Title: The short-chain fatty acid receptors Gpr41/43 regulate bone mass by promoting adipogenic differentiation of mesenchymal stem cells

doi: 10.3389/fendo.2024.1392418

Figure Lengend Snippet: Skeletal phenotype of Gpr41/43 double-deficient mice. (A) Study design: Femoral phenotype of WT (white bars) and Gpr41/43 dKO (blue bars) mice were investigated at 6 (n=5 WT, n=5 Grp41/43 dKO), 13 (n=7 WT, n=5 Gpr41/43 dKO), and 30 (n=3 WT, n=4-5 Gpr41/43 dKO) weeks of age. µCT-based quantification of (B) trabecular bone volume per tissue volume (BV/TV), (C) trabecular numbers (Tb.N), (D) trabecular spacing (Tb.Sp), (E) cortical thickness (Cort.Th), (F) cortical porosity (Cort.Por) and (G) trabecular thickness (Tb.Th). (H) Representative µCT images of femora from 6 13 and 30 weeks old male WT mice showing whole femurs (top, the virtual cut plane appears red), cortical (Ct., middle) and trabecular bone (Tb., bottom). (I) Bone formation marker procollagen type 1 N-terminal propeptide (PINP) and (J) bone turnover marker C-terminal telopeptide (CTX) were measured in blood plasma at indicated time points of age. Data were shown as dot plots with median values indicated as horizontal bars ± SEM analyzed by Student’s t-test. *p < 0.05 vs. Gpr41/43 dKO, **p < 0.01 vs. Gpr41/43 dKO.

Article Snippet: Quantitative real-time PCR was performed with a QuantStudioTM 5 Real-Time PCR System (Thermo Fisher Scientific) using assays-on-demand primer (Applied Biosystems, assay IDs: Acp5: Mm00475698_m1, Clcn7: Mm00442400_m1, Ctsk: Mm00484039_m1, Col1a1 : Mm00801666_g1, Adipoq : Mm00456425_m1, Eno3 : Mm00468267_m1, Gpr41 : Mm02621638_s1, Gpr43 : Mm02620654_s1, Kcnk1 : Mm00492791_m1, Pparg : Mm00440945_m1, Phex : Mm00448119_m1, Ptgs2 : Mm00478374_m1, Lpl : Mm00434764_m1) that were premixed with TaqMan Universal MasterMix II (Applied Biosystems).

Techniques: Marker, Clinical Proteomics

Journal: Frontiers in Endocrinology

Article Title: The short-chain fatty acid receptors Gpr41/43 regulate bone mass by promoting adipogenic differentiation of mesenchymal stem cells

doi: 10.3389/fendo.2024.1392418

Figure Lengend Snippet: Deletion of Gpr41 and Gpr43 increases trabecular bone mass in mice. (A) Study design of histomorphometric analysis from WT (white bars) and Gpr41/43 dKO (blue bars) mice at 6, 13 and 30 weeks of age. (B-D) Histomorphometric evaluation of trabecular bone parameters in tibia. (B) Bone volume per tissue volume, (C) trabecular numbers (Tb.N), (D) trabecular thickness (Tb.Th). Data were shown as mean values ± SEM and analyzed by Student’s t-test. n=3-7 mice per group. *p < 0.05 vs. Gpr41/43 dKO, **p < 0.01 vs. Gpr41/43 dKO. (E) Representative undecalcified histological sections of Von Kossa/van Gieson stained tibia from 6, 13 and 30 weeks old male WT and Gpr41/43 dKO. (F) Representative undecalcified histological sections of vertebral bodies from 6, 13 and 30 weeks old male WT and Gpr41/43 dKO mice. Von Kossa/van Gieson stain. (G-N) Histomorphometric evaluation of trabecular bone parameters in the same sections. (G) Bone volume per tissue volume, (H) trabecular thickness (Tb.Th), (I) trabecular numbers (Tb.N) and (J) trabecular spacing (Tb.Sp). (K-N) Histomorphometric evaluation of cellular bone remodeling parameters in the same sections of vertebral bodies. (K) Number of osteoblasts/bone perimeter (N.Ob/B.Pm), (L) osteoblasts surface per bone surface (Ob.S/BS), (M) number of osteoclasts/bone perimeter (N.Oc/B.Pm), (N) osteoclasts surface per bone surface (Oc.S/BS). Data were shown as dot plots with median values indicated as horizontal bars ± SEM and analyzed by Student’s t-test. n=3-7 mice per group. *p < 0.05 vs. Gpr41/43 dKO, **p < 0.01 vs. Gpr41/43 dKO, ***p < 0.001 vs. Gpr41/43 dKO.

Article Snippet: Quantitative real-time PCR was performed with a QuantStudioTM 5 Real-Time PCR System (Thermo Fisher Scientific) using assays-on-demand primer (Applied Biosystems, assay IDs: Acp5: Mm00475698_m1, Clcn7: Mm00442400_m1, Ctsk: Mm00484039_m1, Col1a1 : Mm00801666_g1, Adipoq : Mm00456425_m1, Eno3 : Mm00468267_m1, Gpr41 : Mm02621638_s1, Gpr43 : Mm02620654_s1, Kcnk1 : Mm00492791_m1, Pparg : Mm00440945_m1, Phex : Mm00448119_m1, Ptgs2 : Mm00478374_m1, Lpl : Mm00434764_m1) that were premixed with TaqMan Universal MasterMix II (Applied Biosystems).

Techniques: Staining

Journal: Frontiers in Endocrinology

Article Title: The short-chain fatty acid receptors Gpr41/43 regulate bone mass by promoting adipogenic differentiation of mesenchymal stem cells

doi: 10.3389/fendo.2024.1392418

Figure Lengend Snippet: Effect of inulin-containing on bone mass in Gpr41/43-defient mice. (A) Study design: 13 weeks old male WT (white bars) and Gpr41/43 dKO mice (blue bars) were fed an inulin-containing diet (ICD) or a respective control diet (CD) for 6 weeks. (B) Average daily food intake, (C) body weight, (D) fat mass relative to body weight (BW), (E) lean mass relative to BW, (F) liver weight. (G) By using GC-MS analysis, levels of SCFAs in portal plasma were analyzed acetate (C2), propionate (C3), butyrate (C4), nd (not defined). (H) Representative µCT images of femora from WT and Gpr41/43 dKO showing whole femurs (top, the virtual cut plane appears red), cortical (Ct., middle) and trabecular bone (Tb., bottom). µCT-based quantification of (I) trabecular bone volume per tissue volume (BV/TV), (J) trabecular numbers (Tb.N), (K) trabecular thickness (Tb.Th), (L) trabecular spacing (Tb.Sp) (M) cortical thickness (Cort.Th), (N) cortical porosity (Cort.Por), (N) ,. Data were shown as dot plots with median values indicated as horizontal bars ± SEM. +++ p < 0.001 WT CD vs. Gpr41/43 dKO CD,*p < 0.05 WT CD vs. WT ICD, **p < 0.01 WT CD vs. WT ICD, ***p < 0.001 WT CD vs. WT ICD, # p < 0.05 Gpr41/43 dKO CD vs Gpr41/43 dKO ICD, ## p < 0.01 Gpr41/43 dKO CD vs Gpr41/43 dKO ICD, § p < 0.05 WT ICD vs Gpr41/43 dKO ICD, §§ p < 0.01 WT ICD vs Gpr41/43 dKO ICD, §§§ p < 0.01 WT ICD vs Gpr41/43 dKO ICD determined by two-way ANOVA. WT, CD: n=3, Gpr41/43, CD: n=3, WT, ICD: n=4, Gpr41/43 dKO, ICD: n=8.

Article Snippet: Quantitative real-time PCR was performed with a QuantStudioTM 5 Real-Time PCR System (Thermo Fisher Scientific) using assays-on-demand primer (Applied Biosystems, assay IDs: Acp5: Mm00475698_m1, Clcn7: Mm00442400_m1, Ctsk: Mm00484039_m1, Col1a1 : Mm00801666_g1, Adipoq : Mm00456425_m1, Eno3 : Mm00468267_m1, Gpr41 : Mm02621638_s1, Gpr43 : Mm02620654_s1, Kcnk1 : Mm00492791_m1, Pparg : Mm00440945_m1, Phex : Mm00448119_m1, Ptgs2 : Mm00478374_m1, Lpl : Mm00434764_m1) that were premixed with TaqMan Universal MasterMix II (Applied Biosystems).

Techniques: Control, Gas Chromatography-Mass Spectrometry, Clinical Proteomics

Journal: Frontiers in Endocrinology

Article Title: The short-chain fatty acid receptors Gpr41/43 regulate bone mass by promoting adipogenic differentiation of mesenchymal stem cells

doi: 10.3389/fendo.2024.1392418

Figure Lengend Snippet: Inulin-containing diet decreases bone marrow adiposity. (A) Study design: 13 weeks old male WT (white bars) and Gpr41/43 dKO mice (blue bars) were fed an inulin-containing diet (ICD) or a respective control diet (CD) for 6 weeks. (B) Representative undecalcified histological sections and of vertebral bodies from 19 weeks old male WT and Gpr41/43 dKO mice fed an inulin-containing diet (ICD) or a respective control diet (CD). (C–F) Histomorphometric evaluation of trabecular bone parameters in the same sections. (C) Bone volume per tissue volume, (D) trabecular numbers (Tb.N), (E) trabecular spacing (Tb.Sp), (F) trabecular thickness (Tb.Th). (G) Representative images of toluidine blue stained tibia sections and red arrows indicate bone marrow adipocytes. (H) Quantification of bone marrow adiposity from the same section. Data were shown as dot plots with median values indicated as horizontal bars ± SEM + p < 0.05 WT CD vs. Gpr41/43 dKO CD, ++ p < 0.01 WT CD vs. Gpr41/43 dKO CD, *p < 0.05 WT CD vs. WT ICD, # p < 0.05 Gpr41/43 dKO CD vs. Gpr41/43 dKO ICD, § p < 0.05 WT ICD vs. Gpr41/43 dKO ICD, determined by two-way ANOVA. WT, CD: n=3, Gpr41/43, CD: n=3, WT, ICD: n=4, Gpr41/43 dKO, ICD: n=8.

Article Snippet: Quantitative real-time PCR was performed with a QuantStudioTM 5 Real-Time PCR System (Thermo Fisher Scientific) using assays-on-demand primer (Applied Biosystems, assay IDs: Acp5: Mm00475698_m1, Clcn7: Mm00442400_m1, Ctsk: Mm00484039_m1, Col1a1 : Mm00801666_g1, Adipoq : Mm00456425_m1, Eno3 : Mm00468267_m1, Gpr41 : Mm02621638_s1, Gpr43 : Mm02620654_s1, Kcnk1 : Mm00492791_m1, Pparg : Mm00440945_m1, Phex : Mm00448119_m1, Ptgs2 : Mm00478374_m1, Lpl : Mm00434764_m1) that were premixed with TaqMan Universal MasterMix II (Applied Biosystems).

Techniques: Control, Staining

Journal: Frontiers in Endocrinology

Article Title: The short-chain fatty acid receptors Gpr41/43 regulate bone mass by promoting adipogenic differentiation of mesenchymal stem cells

doi: 10.3389/fendo.2024.1392418

Figure Lengend Snippet: Acetate facilitates adipocytic differentiation of mesenchymal stem cells. (A-D) Bone marrow cells from tibiae and femora of 8-18-week-old C57Bl/6J mice were isolated and differentiated into osteoclasts. (A) After 12 days of differentiation, osteoclasts were stained for activity of tartrate-resistant acid phosphatase (TRAP). (B-D) Relative gene expression level (fold difference) of indicated osteoclast markers Acp5 , Clcn7 and Ctsk of WT (n=5 white bars) and Gpr41/43 dKO (n=6) bdlue bars) osteoclasts. Data are shown as dot plots with median values indicated as horizontal bars ± SEM. (E–O) Bone marrow from WT mice was isolated and mesenchymal stem cells were cultured with cultivation medium for 7 days followed by cultivation with osteoblastic medium in the absence or presence of acetate (500 µmol) for further 5 days. (E) Alizarin red staining of control (white bar) and acetate-treated WT osteoblasts (red bar). (F) Quantification of extracellular matrix mineralization from control and acetate-treated WT osteoblasts. (G) The expression pattern of downregulated and induced adipocyte and osteoblast marker genes, as determined by Gene Chip hybridization comparing control (n=3 pooled) and acetate-treated cells (n=3 pooled). Gapdh expression was monitored as positive loading control. Data are shown as dot plots with median values indicated as horizontal bars ± SEM. **p < 0.01 determined by Student’s t-test. Control WT osteoblasts n=12, acetate-treated WT osteoblasts n=5. (H-O) Relative gene expression level (fold difference) of indicated osteoblast markers of WT (white bars) and Gpr41/43 dKO mice osteoblasts (red bars) (H-K) Phex , Ptgs2 , Kcnk1 , Col1a1 and (L-O) adipocyte markers Pparg , Lpl , Adipoq , Eno3 . Data are shown as dot plots with median values indicated as horizontal bars ± SEM, and different icons indicate significant differences between groups (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, $$ p < 0.01, $$$ p < 0.001, $$$$ p< 0.0001, # p < 0.05, ## p < 0.01, § p < 0.05) determined by two-way ANOVA. Control WT osteoblasts n=6, acetate-treated WT osteoblasts n=4. Control Gpr41/43 dKO osteoblasts n=6, acetate-treated Gpr41/43 dKO osteoblasts n=6.

Article Snippet: Quantitative real-time PCR was performed with a QuantStudioTM 5 Real-Time PCR System (Thermo Fisher Scientific) using assays-on-demand primer (Applied Biosystems, assay IDs: Acp5: Mm00475698_m1, Clcn7: Mm00442400_m1, Ctsk: Mm00484039_m1, Col1a1 : Mm00801666_g1, Adipoq : Mm00456425_m1, Eno3 : Mm00468267_m1, Gpr41 : Mm02621638_s1, Gpr43 : Mm02620654_s1, Kcnk1 : Mm00492791_m1, Pparg : Mm00440945_m1, Phex : Mm00448119_m1, Ptgs2 : Mm00478374_m1, Lpl : Mm00434764_m1) that were premixed with TaqMan Universal MasterMix II (Applied Biosystems).

Techniques: Isolation, Staining, Activity Assay, Gene Expression, Cell Culture, Control, Expressing, Marker, Hybridization

Journal: Frontiers in Endocrinology

Article Title: The short-chain fatty acid receptors Gpr41/43 regulate bone mass by promoting adipogenic differentiation of mesenchymal stem cells

doi: 10.3389/fendo.2024.1392418

Figure Lengend Snippet: Graphical abstract showing the effect of acetate on WT and Gpr41/43 deficient mesenchymal stem cells (MSC). In the absent of short chain fatty receptors Gpr41 and Gpr43 treatment of MSC with acetate gives rise toward a greater extent osteoblastic differentiation compared to adipocytic differentiation.

Article Snippet: Quantitative real-time PCR was performed with a QuantStudioTM 5 Real-Time PCR System (Thermo Fisher Scientific) using assays-on-demand primer (Applied Biosystems, assay IDs: Acp5: Mm00475698_m1, Clcn7: Mm00442400_m1, Ctsk: Mm00484039_m1, Col1a1 : Mm00801666_g1, Adipoq : Mm00456425_m1, Eno3 : Mm00468267_m1, Gpr41 : Mm02621638_s1, Gpr43 : Mm02620654_s1, Kcnk1 : Mm00492791_m1, Pparg : Mm00440945_m1, Phex : Mm00448119_m1, Ptgs2 : Mm00478374_m1, Lpl : Mm00434764_m1) that were premixed with TaqMan Universal MasterMix II (Applied Biosystems).

Techniques:

Journal: Scientific Reports

Article Title: Butyrate produced by gut commensal bacteria activates TGF-beta1 expression through the transcription factor SP1 in human intestinal epithelial cells

doi: 10.1038/s41598-018-28048-y

Figure Lengend Snippet: Butyrate mediated impact on TGFB1 is independent of GPR41, GPR43, GPR109a receptors and MCT1 transporter. ( A ) HT-29-TGF prom reporter cells were incubated for 24 h with selective GPR agonists: GPR41: AR420626 (1 μM) and 1-MCPC (1 mM); GPR43: 4-CMTB (1 μM) and Tiglic acid (1 mM); GPR109a: Niacin (1 mM) and MK1903 (1 μM) or with DMSO (vehicle) or butyrate (2 mM). ( B ) HT-29-TGF prom reporter cells were incubated with GPRs sub-unit inhibitors: Pertussis toxin 2 h before (Ptx, 0.2 μg/ml) and U73122 (10 μM) prior butyrate 2 mM stimulation and left for a total 24 h of incubation. ( C ), HT-29-TGF prom reporter cells were incubated for 24 h with selective MCT1 inhibitors: pCMB (100 μM) and AR-C155858 (400 nM),. TGFB1 expression was measured by luciferase activity and expressed as median ± quartiles of fold change towards un-stimulated cells. N ≥ 3, Wilcoxon test, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The following Taqman Gene expression assay probes were used: GAPDH Hs02758991_g1, GPR43 Hs00271142_s1, GPR41 Hs02519193_g1, GPR109a Hs02341584_s1, SMCT1 Hs00377618_m1, and MCT1 Hs01585687_m1.

Techniques: Incubation, Expressing, Luciferase, Activity Assay