Journal: International Journal of Molecular Medicine

Article Title: The interplay of BMP4 and IL-7 regulates the apoptosis of intestinal intraepithelial lymphocytes under conditions of ischemia/reperfusion

doi: 10.3892/ijmm.2018.3480

Figure Lengend Snippet: The bone morphogenetic protein (BMP) signaling pathway is activated in intestinal epithelial cells and intraepithelial lymphocytes (IELs) following ischemia/reperfusion (I/R). (A) The level of BMP4 protein (red) expression significantly increased in the mid-to-distal villus region after 6 h of I/R, according to immunofluorescence staining. (B) The expression levels of the type I BMP receptor and phosphorylated nuclear factor (p-NF)-κB were both significantly increased after 6 h of I/R in IELs. * P<0.05, 3-4 mice/group; the results are representative of three independent experiments.

Article Snippet: Recombinant BMP4 protein (cat. no. 315-27; 30 ng/ml) and recombinant murine IL-7 protein (cat. no. 217-17; 2 ng/ml) were both purchased from ProteinTech Group, Inc. Pyrrolidine dithiocarbamate (PDTC; cat. no. S1808; 30 ng/ml), an inhibitor of NF-κB, was purchased from Beyotime Institute of Biotechnology (Wuhan, China).

Techniques: Expressing, Immunofluorescence, Staining

Journal: International Journal of Molecular Medicine

Article Title: The interplay of BMP4 and IL-7 regulates the apoptosis of intestinal intraepithelial lymphocytes under conditions of ischemia/reperfusion

doi: 10.3892/ijmm.2018.3480

Figure Lengend Snippet: Activation of nuclear factor (NF)-κB signaling by bone morphogenetic protein (BMP)4 stimulation in intraepithelial lymphocytes (IELs). Flow cytometry determined the expression of the BMP type I receptor and phosphorylated NF-κB in IELs in culture. Following treatment with BMP4 for 6 h, the expression of the BMP type I receptor and phosphorylated NF-κB was significantly increased compared with that in the control group. NOGGIN partially decreased the expression of the BMP type I receptor, as well as NF-κB transcriptional activity. The results are representative of three independent experiments. P<0.05.

Article Snippet: Recombinant BMP4 protein (cat. no. 315-27; 30 ng/ml) and recombinant murine IL-7 protein (cat. no. 217-17; 2 ng/ml) were both purchased from ProteinTech Group, Inc. Pyrrolidine dithiocarbamate (PDTC; cat. no. S1808; 30 ng/ml), an inhibitor of NF-κB, was purchased from Beyotime Institute of Biotechnology (Wuhan, China).

Techniques: Activation Assay, Flow Cytometry, Expressing, Control, Activity Assay

Journal: International Journal of Molecular Medicine

Article Title: The interplay of BMP4 and IL-7 regulates the apoptosis of intestinal intraepithelial lymphocytes under conditions of ischemia/reperfusion

doi: 10.3892/ijmm.2018.3480

Figure Lengend Snippet: Bone morphogenetic protein (BMP)4 induces intraepithelial lymphocytes (IELs) to undergo apoptosis. Intestinal IELs were examined by flow cytometry for markers of apoptosis (FITC-Annexin V and PI). FITC-Annexin V + /PI + indicates late apoptosis, FITC-Annexin V + /PI − indicates early apoptosis, and FITC-Annexin V − /PI − indicates live cells. The extent of apoptosis of IELs after treatment with BMP4 for 6 h was then determined. The expression of FITC-Annexin V + /PI + IELs in the BMP4 group was significantly higher compared with that in the control group, but these effects were decreased by treatment with NOGGIN or pyrrolidine dithiocarbamate (PDTC). P<0.05, 3-4 mice/group; each experiment was repeated three times.

Article Snippet: Recombinant BMP4 protein (cat. no. 315-27; 30 ng/ml) and recombinant murine IL-7 protein (cat. no. 217-17; 2 ng/ml) were both purchased from ProteinTech Group, Inc. Pyrrolidine dithiocarbamate (PDTC; cat. no. S1808; 30 ng/ml), an inhibitor of NF-κB, was purchased from Beyotime Institute of Biotechnology (Wuhan, China).

Techniques: Flow Cytometry, Expressing, Control

Journal: International Journal of Molecular Medicine

Article Title: The interplay of BMP4 and IL-7 regulates the apoptosis of intestinal intraepithelial lymphocytes under conditions of ischemia/reperfusion

doi: 10.3892/ijmm.2018.3480

Figure Lengend Snippet: Inhibition of bone morphogenetic protein (BMP)4 induces the apoptosis of intraepithelial lymphocytes (IELs) that has been stimulated by interleukin (IL)-7. Flow cytometry and apoptosis markers (FITC-Annexin V and PI) were used to examine IEL apoptosis after treatment with BMP4, IL-7 or BMP4 + IL-7 for 6 h. The expression of FITC-Annexin V + /PI + IELs in the IL-7 treatment group was significantly lower compared with that in the control group, whereas the expression of FITC-Annexin V + /PI + IELs in the BMP4 treatment group was significantly higher compared with that in the control group. However, the expression of FITC-Annexin V + /PI + IELs in the BMP4 + IL-7 treatment group exhibited no changes compared with the control group. Each experiment was repeated three times; 4-5 mice/group. P<0.05.

Article Snippet: Recombinant BMP4 protein (cat. no. 315-27; 30 ng/ml) and recombinant murine IL-7 protein (cat. no. 217-17; 2 ng/ml) were both purchased from ProteinTech Group, Inc. Pyrrolidine dithiocarbamate (PDTC; cat. no. S1808; 30 ng/ml), an inhibitor of NF-κB, was purchased from Beyotime Institute of Biotechnology (Wuhan, China).

Techniques: Inhibition, Flow Cytometry, Expressing, Control

Journal: International Journal of Molecular Medicine

Article Title: The interplay of BMP4 and IL-7 regulates the apoptosis of intestinal intraepithelial lymphocytes under conditions of ischemia/reperfusion

doi: 10.3892/ijmm.2018.3480

Figure Lengend Snippet: Interleukin (IL)-7 downregulates the bone morphogenetic protein (BMP) signaling pathway in intestinal epithelial cells (IECs) and intraepithelial lymphocytes (IELs). (A) Western blot analysis was used to determine the expression of BMP4 in IEC-6 cells following treatment with IL-7 for 6 h. The expression of BMP4 was significantly decreased compared with that in the control group. (B) Flow cytometry, was used to detect the phosphorylation of nuclear factor (NF)-κB following treatment with IL-7 for 6 h. The expression of phosphorylated NF-κB was significantly decreased compared with that in the control group. Each experiment was repeated three times; 4-5 mice/group. * P<0.05 vs. control. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Recombinant BMP4 protein (cat. no. 315-27; 30 ng/ml) and recombinant murine IL-7 protein (cat. no. 217-17; 2 ng/ml) were both purchased from ProteinTech Group, Inc. Pyrrolidine dithiocarbamate (PDTC; cat. no. S1808; 30 ng/ml), an inhibitor of NF-κB, was purchased from Beyotime Institute of Biotechnology (Wuhan, China).

Techniques: Western Blot, Expressing, Control, Flow Cytometry, Phospho-proteomics

Journal: International Journal of Molecular Medicine

Article Title: The interplay of BMP4 and IL-7 regulates the apoptosis of intestinal intraepithelial lymphocytes under conditions of ischemia/reperfusion

doi: 10.3892/ijmm.2018.3480

Figure Lengend Snippet: Bone morphogenetic protein (BMP)4 regulates the interleukin (IL)-7α/CD127 signaling pathway in intestinal epithelial cells (IECs) and intraepithelial lymphocytes (IELs). (A) Western blot analysis determined the expression of IL-7 in IEC-6 cells following treatment with BMP4 for 6 h. The IL-7 level was significantly decreased compared with that in the control group. (B) Flow cytometry, was used to detect the expression of CD127 and phosphorylated signal transducer and activator of transcription (STAT)5 proteins following treatment with BMP4 for 6 h. The levels of CD127 and phosphorylated STAT5 proteins were significantly decreased compared with those in the control group. Each experiment was repeated three times; 4-5 mice/group. * P<0.05 for control vs. treatment with BMP4 alone; ** P<0.05 for control vs. the BMP4 + NOGGIN group. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Recombinant BMP4 protein (cat. no. 315-27; 30 ng/ml) and recombinant murine IL-7 protein (cat. no. 217-17; 2 ng/ml) were both purchased from ProteinTech Group, Inc. Pyrrolidine dithiocarbamate (PDTC; cat. no. S1808; 30 ng/ml), an inhibitor of NF-κB, was purchased from Beyotime Institute of Biotechnology (Wuhan, China).

Techniques: Western Blot, Expressing, Control, Flow Cytometry

Journal: eLife

Article Title: Evaluation of Gremlin-1 as a therapeutic target in metabolic dysfunction-associated steatohepatitis

doi: 10.7554/eLife.95185

Figure Lengend Snippet: ( A ) Gremlin-1/BMP4 inhibition enzyme linked immunosorbent assay (ELISA), measuring aG1-Ab ability to inhibit Gremlin-1 binding to BMP4. Higher absorbance indicates more Gremlin-1 binding to BMP4. IC50=2.7–3.1×10 –9 M. Dots and error bars represent mean ± SD and lines show fitted four-parameter log-logistic curve. ( B ) C2C12 BMP-responsive element Luc reporter gene assay. Luminescence is plotted over response to serial dilutions of anti-Gremlin-1 antibodies with higher luminescence indicating increased BMP4 activity. Dots and error bars represent mean ± SD and lines show fitted four-parameter log-logistic curve. EC50=1.27–1.36 × 10 –8 M. ( C ) SMAD1 phosphorylation on LX-2 cells treated with either BMP4, BMP4 and Gremlin-1 or BMP4, Gremlin-1 and serial dilutions of therapeutic antibody. Dots and error bars represent mean ± SD and lines show fitted four-parameter log-logistic curve. K D [’0032]=2.04 nM, K D [’0030]=3.96 nM. ( D ) Size exclusion chromatography for Gremlin-1 in combination with heparin-displacing (’0030) or non-heparin-displacing (’0032) anti-Gremlin-1 antibody. The graph shows UV signal (continuous line) and estimated molar mass (points) on the y-axis depending on the eluting volume on the x-axis. Text annotations give the estimated molar mass corresponding to each peak. ( E ) Fluorescence polarisation heparin-binding assay. Serial dilutions of Gremlin-1 were incubated with fixed amounts of fluorescein-heparan sulfate and 1.5-fold molar excess anti-Gremlin-1 antibody. Increased fluorescence indicates reduced mobility of heparin molecules. Dots and error bars represent mean ± SD and lines show fitted four-parameter log-logistic curve. K D [Grem1]=13.54 nM, K D [’0032]=19.56 nM and K D [’0030]=118.65 nM. ( F ) Gremlin-1 cell association assay. The upper panel shows a confocal view and the lower panel a three-dimensional cell surface view for Atto-532-labelled Gremlin-1 (yellow) on LX-2 cells (labelled with CellMask Blue). Representative images for different combinations of 250 nM Gremlin-1 and isotype or anti-Gremlin-1 antibodies are given. BMP, bone morphogenetic protein. Figure 3—source data 1. Excel spreadsheet containing data displayed in panels A–E.

Article Snippet: Thereafter, the anti-Gremlin/Gremlin-1 mixes were pre-incubated with 0.1 nM BMP4 (recombinant human, R&D Systems, #314-BP/CF) for 10 min at room temperature on a plate shaker at 350 RPM.

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Binding Assay, Reporter Gene Assay, Activity Assay, Size-exclusion Chromatography, Fluorescence, Incubation, Histone Association Assay

Journal: Redox Biology

Article Title: Endothelial TET2 regulates the white adipose browning and metabolism via fatty acid oxidation in obesity

doi: 10.1016/j.redox.2023.103013

Figure Lengend Snippet: Endothelial TET2 modulates lipolysis through secretion of BMP4. A , Diagram of in vivo WAT explants assay. B and C , FFA level of SAT (B) and VAT (C) obtained from WT or TET2 EC−KO mice at the time of 0 min or 120 min (n = 6 mice/group). D , Diagram of co-culture of bEnd.3 and adipocytes. E , qPCR analysis of bEnd.3 after overexpressing TET2 by adenovirus. n = 3/group. F, Representative images of oil red O staining in adipocytes after co-culturing with TET2 overexpressed bEnd.3. Scale bars = 50 μm. G , qPCR analysis of lipolysis-related genes in adipocytes after co-culturing with TET2 overexpressed bEnd.3. n = 3/group. H , Cell culture supernatants of HUVECs transfected with ctrl siRNA or TET2 siRNA were detected by human adipokine array kit. I , Relative mean pixel density of H was calculated. n = 3/group. J , Relative mRNA levels of BMP4 in HUVECs transfected with Ctrl siRNA or TET2 siRNA. n = 3/group. K , Serum BMP4 level of WT and TET2 EC−KO mice with HFD for 16 weeks. n = 6 mice/group. L , Representative images of adipocyte morphology (H&E), microvessels (IB4, red), UCP1 staining in SAT from WT or TET2 EC−KO mice with or without local injection of BMP4 in CL316243-treated model. M , Quantification of L. n = 6 mice/group. All data are mean ± SEM. E, G, I, J, K, unpaired Student's t -test. B, C, M, Two-way ANOVA with Bonferroni post hoc test. * P < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Insulin, BMP4 and global DNA methylation levels were measured by murine INS ELISA kits (Cat#YCQZ-10385, YC Bio), murine BMP4 ELISA kits (Cat#EK0316, Boster), and Global DNA Methylation–LINE-1 Kit (Cat#55017, Active Motif) respectively.

Techniques: In Vivo, Co-Culture Assay, Staining, Cell Culture, Transfection, Injection

Journal: Redox Biology

Article Title: Endothelial TET2 regulates the white adipose browning and metabolism via fatty acid oxidation in obesity

doi: 10.1016/j.redox.2023.103013

Figure Lengend Snippet: NRF2 recruits TET2 to regulate FAO. A , Binding sites of TET2 and NRF2 were predicted using Gramm-X. Blue, TET2; Cyan, NRF2; Red, binding sites. B and C , Lysate of HUVECs was immunoprecipitated with anti-TET2 antibody (B) or anti-NRF2 antibody (C) and then immunoblotted with the indicated antibodies. D , Schematic illustration of different plasmids of TET2 and NRF2. E and F , TET2-His and NRF2-Flag were ectopically expressed in HEK293T and the lysate of HEK293T was immunoprecipitated with anti-His (E) antibody or anti-Flag antibody (F). G , NRF2 was ectopically expressed with different domains of TET2 HEK293T and the lysate of HEK293T was immunoprecipitated with anti-Flag antibody. H , The 5-mC levels of HUVECs transfected with or without TET2 siRNA. n = 3/group. I , The methylation status of BMP4 and CPT1A promoter in HUVECs treated with AdNC or AdTET2. J , Predict binding sites of NRF2 in the promoters of CPT1A and BMP4 by JASPAR. K . qPCR analysis of the mRNA expression levels of CPT1A and BMP4 in HUVECs infected with TET2 adenovirus in presence or absence of NRF2 siRNA. n = 3/group. L . qPCR analysis of the mRNA expression levels of CPT1A and BMP4 in HUVECs treated with bardoxolone methyl in presence or absence of TET2 siRNA. n = 3/group. All data are mean ± SEM. E, Two-way ANOVA with Bonferroni post hoc test. * P < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Insulin, BMP4 and global DNA methylation levels were measured by murine INS ELISA kits (Cat#YCQZ-10385, YC Bio), murine BMP4 ELISA kits (Cat#EK0316, Boster), and Global DNA Methylation–LINE-1 Kit (Cat#55017, Active Motif) respectively.

Techniques: Binding Assay, Immunoprecipitation, Transfection, Methylation, Expressing, Infection