Journal: Frontiers in Immunology

Article Title: Electroacupuncture alleviates pain by activating the MD2/TLR4/NF-κB pathway in the ST36 acupoint

doi: 10.3389/fimmu.2025.1626755

Figure Lengend Snippet: Functional enrichment analysis and distribution of differentially expressed proteins in the spinal cord of mice treated with EA and MD2 knockdown. (A) PCA analysis of all samples, each point denotes a sample, and different colors indicate different groups. (B) Each color represents a group, and the number of differentially expressed genes is indicated in the figure. (C) The hierarchical clustering results are presented as a dendrogram heatmap, with the x-axis and y-axis corresponding to samples and differentially expressed proteins, respectively. In the heatmap, red indicates significant upregulation, blue indicates significant downregulation, and gray represents the absence of quantitative data. (D, E) The vertical axis represents the GO level 2 functional annotation information, including molecular function, cellular component, and biological process. The horizontal axis indicates the significance of enrichment, with larger values on the horizontal axis representing a higher significance level of the corresponding GO functions. (F, G) KEGG pathway analysis of differential proteins. The vertical axis represents the names of pathways involving differentially expressed proteins. The horizontal axis indicates the significance of enrichment, with larger values signifying a higher level of significance for the enrichment in the corresponding pathway. The color gradient represents the magnitude of the enrichment factor (Rich Factor ≤ 1), with colors closer to red indicating a larger Rich Factor value. (H) Circos plot of the relationship between the significantly enriched KEGG pathway and the protein. (I) Bar chart showing the expression levels of the 5 key differentially expressed proteins. (J–L) The expression level of Grem1 (J) , BMP4 (K) , COX2 (L) (n = 3), ** P < 0.01 vs. the AIA+LVcon, # P < 0.01 vs. the AIA+LVcon+EA, ## P < 0.01 vs. the AIA+LVcon+EA.

Article Snippet: The blots were then washed and incubated overnight at 4°C with anti-MD2 antibody (1:1000, sc-80183, Santa, USA; 1:1000, ab24182, abcam, UK), anti-TLR4 antibody (1:1000, sc-293072, Santa, USA; 1:1000, 19811-1-AP, Proteintech Group, China), anti-P-P65 antibody (1:1000, 3033S, Cell Signaling Technology, USA), anti-P65 antibody (1:8000, 8242, Cell Signaling Technology, USA), anti-Grem1 antibody (1:800, sc-293426, Santa, USA), anti-BMP4 antibody (1:200, 12492-1-AP, Proteintech Group, Inc, USA), anti-COX2 antibody (1:500, A3560, ABclonal, China), anti-GAPDH antibody (1:3000000, A19056, ABclonal, China).

Techniques: Functional Assay, Knockdown, Expressing

Journal: Stem cells international

Article Title: Developing a Novel and Convenient Model for Investigating Sweat Gland Morphogenesis from Epidermal Stem Cells.

doi: 10.1155/2019/4254759

Figure Lengend Snippet: Figure 4: Detection of key soluble morphogens in the system of sweat gland morphogenesis and the impact of BMP receptor inhibitor and EGF receptor inhibitor on sweat gland development in vitro. BMP4 and EGF demonstrated sharped difference in the medium of the sweat gland organogenesis system. BMP receptor inhibitor could block the formation of sweat gland in this system, while EGF receptor inhibitor significantly reduced the expression of K18. (a) The variation tendency of BMP4 and EGF in the medium of system. The error bar meant the standard error of BMP4 and EGF concentrations in different systems at different time points. (b) The impact of BMP receptor inhibitor and EFG receptor inhibitor on sweat gland morphogenesis. In comparison with the control group, EGF receptor inhibitor significantly reduced the expression of K18, but glandular structure was still observed. BMP receptor inhibitor completely blocks the expression of K18, and no glandular structure was observed. All the nuclei were counterstained with DAPI (DAPI: blue; K18: red; bars = 200 μm and 50 μm; K18: cytokeratin 18; IM: light microscope; H&E: hematoxylin-eosin staining; IF: immunofluorescence staining).

Article Snippet: BMP4 and EGF concentrations in supernatant were measured using a mouse BMP4 (CSB-E04512m, Cusabio, Wuhan, Hubei, China) and EGF ELISA kit (EM014-96, ExCell Bio, Shanghai, China).

Techniques: In Vitro, Blocking Assay, Expressing, Comparison, Control, Light Microscopy, Staining

Journal: The FASEB Journal

Article Title: Low‐Intensity Pulsed Ultrasound Promotes Osteogenesis in Porous Titanium Alloys Through miR ‐1187/ BMP4 Pathway

doi: 10.1096/fj.202403395RR

Figure Lengend Snippet: miR‐1187 directly targets BMP4. (A) The predicted binding site between miR‐1187 and BMP4 by bioinformatics analysis. (B) The luciferase activity of the BMP4‐WT and BMP4‐MUT in MC3T3‐E1 cells treated with miR‐1187 mimics or NC. (C) mRNA expression of Bmp4 was significantly downregulated in the miR‐1187 mimics‐transfected group. (D) Western blot analysis for the expression of BMP4 protein in miR‐1187 mimics and inhibitor‐transfected MC3T3‐E1 cells on Pti. (E) ELISA assay analysis for the expression of supernatant BMP4 secreted protein in miR‐1187 mimics and inhibitor‐transfected MC3T3‐E1cells on Pti. All values represent means ± SD.( n = 3). * p < 0.05, ** p < 0.01.

Article Snippet: BMP4 protein levels in the supernatant were quantified using a mouse BMP‐4 ELISA kit (EK0316, Boster, China) following the manufacturer's protocol.

Techniques: Binding Assay, Luciferase, Activity Assay, Expressing, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: The FASEB Journal

Article Title: Low‐Intensity Pulsed Ultrasound Promotes Osteogenesis in Porous Titanium Alloys Through miR ‐1187/ BMP4 Pathway

doi: 10.1096/fj.202403395RR

Figure Lengend Snippet: BMP4 positively regulates osteogenic differentiation in MC3T3‐E1 cells. (A) Level of BMP4 decreased after transfected with different siRNA‐BMP4, si‐BMP4‐785 was identified to have the highest transfection efficiency. (B) BMP4 transcript levels was determined by RT‐PCR after transfected with overexpression plasmids of BMP4. (C) qRT‐PCR analysis. Level of Col‐1 , Alp , Runx2 , and Ocn mRNA expression was significantly downregulated in BMP4 silencing. (D) qRT‐PCR analysis. Level of Col‐1 , Alp , Runx2 , and Ocn mRNA expression was significantly upregulated in BMP4 overexpression. (E) Western blotting. Expression of BMP4 protein and osteogenesis‐related proteins COL‐1, ALP, RUNX2 as well as statistical analysis after transfecting si‐BMP4. (F) Western blotting. Expression of BMP4 protein and osteogenesis‐related proteins COL‐1, ALP, RUNX2 as well as statistical analysis after transfecting pCDNA3.1‐BMP4. (G) ALP activity detection on day 7 in si‐BMP4 and pCDNA3.1‐BMP4 transfected MC3T3‐E1cells. (H) ALP staining on day 10 in si‐BMP4 and pCDNA3.1‐BMP4 transfected MC3T3‐E1cells (scale bar = 500 μm). (I) Alizarin red S staining and quantitative analysis of Alizarin Red S accumulation on day 21 in si‐BMP4 and pCDNA3.1‐BMP4 transfected MC3T3‐E1cells (scale bar = 200 μm/100 μm), the black arrow indicate the magnified area, the magnified pictures were marked within the square frame in the image. All values represent means ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: BMP4 protein levels in the supernatant were quantified using a mouse BMP‐4 ELISA kit (EK0316, Boster, China) following the manufacturer's protocol.

Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Over Expression, Quantitative RT-PCR, Expressing, Western Blot, Activity Assay, Staining

Journal: The FASEB Journal

Article Title: Low‐Intensity Pulsed Ultrasound Promotes Osteogenesis in Porous Titanium Alloys Through miR ‐1187/ BMP4 Pathway

doi: 10.1096/fj.202403395RR

Figure Lengend Snippet: LIPUS promotes osteogenic differentiation of MC3T3‐E1 through inhibiting miR‐1187 and upregulating BMP4. (A) qRT‐PCR analysis. Effect of daily LIPUS stimulation on mRNA expression of Bmp4. (B) ELISA. Supernatant BMP4 secreted by cultured MC3T3‐E1 cells was determined by ELISA assay. (C) Western blotting. BMP4 level was analyzed by western blot after 7 days LIPUS treatment. (D) Expression of osteogenesis‐related mRNA was analyzed by qRT‐PCR after si‐BMP4 or si‐bmp4 + LIPUS treatment. (E) Expression of osteogenesis‐related protein was analyzed by western blot after si‐BMP4 or si‐BMP4 + LIPUS treatment. (F) ALP activity detection on day 7 in si‐BMP4 and si‐BMP4 + LIPUS‐treated MC3T3‐E1 cells. (G) Bmp4 and osteogenesis‐related genes Col‐1, Alp, Runx2, and Ocn expression in MC3T3‐E1 cells on Pti as indicated treatment by qRT‐PCR. (H) ALP activity in MC3T3‐E1 cells on Pti on day 7 as indicated treatment. (I) Expression of BMP4 and osteogenesis‐related proteins including COL‐1, ALP, RUNX2 in MC3T3‐E1 cells on Pti as indicated treatment by Western blot analysis. All values represent means ± SD ( n = 3). Expression of osteogenesis‐related protein was analyzed by western blot (E1) and quantitative analysis (E2) after si‐BMP4, LIPUS or si‐BMP4+LIPUS treated. Expression of BMP4 and osteogenesis‐ related proteins including COL‐ 1, ALP, RUNX2 in MC3T3‐ E1 cells on Pti as indicated treatment by Western blot analysis(I)andquantitative analysis (J). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: BMP4 protein levels in the supernatant were quantified using a mouse BMP‐4 ELISA kit (EK0316, Boster, China) following the manufacturer's protocol.

Techniques: Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Western Blot, Activity Assay

Journal: The FASEB Journal

Article Title: Low‐Intensity Pulsed Ultrasound Promotes Osteogenesis in Porous Titanium Alloys Through miR ‐1187/ BMP4 Pathway

doi: 10.1096/fj.202403395RR

Figure Lengend Snippet: LIPUS regulates bone formation in vivo. (A) Study plan of the vitro study. (B) qRT‐PCR analysis of mRNA expression of Bmp4 as well as other osteogenesis‐related gene in the de novo bone of different groups. B1: MRNA expression of Bmp4; B2, B3: Osteogenesis‐related gene including Col‐1, Alp, Runx2, and Ocn at 4 weeks and 8 weeks respectively. (C) Micro‐CT analysis. C1: 3D reconstruction of the bone defect area at week 4 and 8. More bone ingrowth is observed as time increases in each group. The white part is the scaffold and the red part is the bone tissue. C2: The POF values for the Pti at 4 and 8 weeks. The POF values differ significantly between the LIPUS and control group, between the si‐BMP4 and control group, as well as between the si‐BMP4 and si‐BMP4 + LIPUS group. (D) D1: The representative merged images of fluorescent double labeling of calcein(green color) and xylenol orange(orange color). The red square indicate the magnified area, the magnified pictures were marked within the white square frame in the image. (scale bar = 200 μm/100 μm). D2: The MAR of the four groups at 4 and 8 weeks. All values represent means ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: BMP4 protein levels in the supernatant were quantified using a mouse BMP‐4 ELISA kit (EK0316, Boster, China) following the manufacturer's protocol.

Techniques: In Vivo, Quantitative RT-PCR, Expressing, Micro-CT, Control, Labeling

Journal: The FASEB Journal

Article Title: Low‐Intensity Pulsed Ultrasound Promotes Osteogenesis in Porous Titanium Alloys Through miR ‐1187/ BMP4 Pathway

doi: 10.1096/fj.202403395RR

Figure Lengend Snippet: The schematic of LIPUS promotes osteogenesis of porous titanium alloys through inhibiting miR‐1187 and upregulating BMP4.

Article Snippet: BMP4 protein levels in the supernatant were quantified using a mouse BMP‐4 ELISA kit (EK0316, Boster, China) following the manufacturer's protocol.

Techniques:

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 BMP4 mRNA and BMP4 expression of MSCs in vitro. (a) Photomicrograph showing positive signal of hybridization is shown as deep brown colour within the cytoplasm in situ hybridization for BMP4 mRNA (arrow, the position where brown colour was expressed at cell cytoplasm was positive, and with haematoxylin counter stain, original magnification ×200) and BMP4‐positive cell cytoplasm were in green (white arrow, the position where green colour was expressed at cell cytoplasm was positive, the position where green colour was not expressed at cell cytoplasm was negative, and with PI counter stain, original magnification ×200), MSCs in control group showed a little BMP4 mRNA and BMP4‐positive staining, PTE treated MSCs show more strong BMP4 mRNA and BMP4‐positive staining. (b) Comparison of the percentages of BMP4 mRNA and BMP4‐positive cell among the four experimental groups after PTE treatment of 3 days. (c) Representative real‐time reverse transcription‐polymerase chain reaction demonstrating an increase in BMP4 mRNA isolated from MSC in response to a different dose of PTE (30 and 300 µg/ml culture media) for 1, 3 and 5 days. (d) Representative ELISA for BMP4 of MSCs cultured increased with increasing time and dose of PTE. *P < 0.05; **P < 0.01 compared to control.

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: Expressing, In Vitro, Hybridization, In Situ Hybridization, Staining, Control, Comparison, Reverse Transcription, Polymerase Chain Reaction, Isolation, Enzyme-linked Immunosorbent Assay, Cell Culture