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: Cell Proliferation

Article Title: Extracts from plastrum testudinis promote proliferation of rat bone‐marrow‐derived mesenchymal stem cells

doi: 10.1111/j.1365-2184.2007.00431.x

Figure Lengend Snippet: Effect of PTE on the cell cycle of MSCs in vitro and in vivo. (a) Representative cell cycle analysis for PI of MSCs cultured treatment with a different dose of PTE (30 µg/ml, 300 µg/ml) for 1 day and 3 days. PI levels of MSCs cultured increased with increasing dose and time of PTE. (b) Representative cell cycle analysis for PI of MSCs isolated from rats that were administered with different dose of PTE (3 mg/kg/day, 30 mg/kg/day) for 1 and 3 days in vivo. The PT levels of MSCs cultured increased with increasing dose and time of PTE. PTE strongly promoted increasing of PI of MSCs in vivo in time‐ and dose‐dependent manner. *P < 0.05; **P < 0.01 compared to control at the same time point. ***P < 0.05; ****P < 0.01 PI levels from 3 days MSCs cultured with and without PTE were compared to those from 1 day MSCs cultured with or without PTE. (c) Detection of CD44 expression by flow cytometry. MSCs isolated from rats that were administered with and without PTE for 3 days in vivo were analysed for expression of CD44 by flow cytometry, negative control represent MSCs incubated with FITC‐conjugated antibody alone. Vertical lines represent maximum fluorescence intensity for FITC‐conjugated antibody. CD44 expression of MSCs isolated from rats that were administered with PTE increased in dose‐dependent manner.

Article Snippet: CD44, probes of BMP4 mRNA, digoxigenin‐labelled probe detection kit, and diaminobenzidine were purchased from Wuhan Boster Biological Technology Co., Ltd. (Wuhan, China); chemicals such as dimethyl sulphoxide (DMSO) and other reagents were also obtained from Sigma.

Techniques: In Vitro, In Vivo, Cell Cycle Assay, Cell Culture, Isolation, Control, Expressing, Flow Cytometry, Negative Control, Incubation, Fluorescence

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