human elisa kit quantikine  (R&D Systems)

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: Frontiers in Cell and Developmental Biology

Article Title: Integrating scRNA-seq and bulk RNA-seq to explore the differentiation mechanism of human nail stem cells mediated by onychofibroblasts

doi: 10.3389/fcell.2024.1416780

Figure Lengend Snippet: Expression of BMP4 in human onychofibroblasts. (A) Major cell types in the human nail unit revealed by scRNA-seq. KC, keratinocyte; FIB, fibroblast; MFLC, myofibroblast-like cell; VEC, vascular endothelial cell; LEC, lymphatic endothelial cell; IMM, immune cell; NC, neural cell; MEL, melanocyte. (B) UMAP visualization of the human OF population. (C) Venn diagram illustrating the number of overlapping DEGs upregulated in OFs between scRNA-seq and bulk RNA-seq. (D) Relative mRNA expression of BMP4 and FGF10 in DF and OF groups. (E) Immunofluorescence staining and (F) MFI of BMP4 in two groups. MFI, Mean Fluorescence Intensity. (G) Western blotting and (H) quantitative analysis of BMP4 in two groups. (I) ELISA of BMP4 in the supernatants of cell cultures from two groups. Scale bars, 100 μm. The results presented are the mean ± SD of six biologically independent replicates. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Cells were seeded in 6-well plates at a density of 2 × 10 5 cells for 72 h. Then the cell supernatant from different samples was collected and analyzed using a human BMP4 ELISA kit (R&D Systems) following the manufacturer’s protocol.

Techniques: Expressing, RNA Sequencing Assay, Immunofluorescence, Staining, Fluorescence, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Integrating scRNA-seq and bulk RNA-seq to explore the differentiation mechanism of human nail stem cells mediated by onychofibroblasts

doi: 10.3389/fcell.2024.1416780

Figure Lengend Snippet: BMP4 derived from human onychofibroblasts regulates differentiation of nail stem cells. (A) Identity, marker genes, and cell proportions of human nail keratinocyte subtypes. (B) In vitro culture showing cell expansion and colony formation of human NSCs. (C) Flow cytometric analysis of KRT15 and KRT1 expression in human NSCs cultured in vitro . (D) Grouping of indirect co-cultures of OFs and NSCs. (E) Relative mRNA expression of stem and differentiation marker genes in keratinocytes under different co-culture conditions. (F) Immunofluorescence staining and (G) MFI of KRT15 and IGFBP7 in three groups. MFI, Mean Fluorescence Intensity. KD , knockdown . Scale bars, 200 μm. The results presented are the mean ± SD of three biologically independent replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, ns, no significant.

Article Snippet: Cells were seeded in 6-well plates at a density of 2 × 10 5 cells for 72 h. Then the cell supernatant from different samples was collected and analyzed using a human BMP4 ELISA kit (R&D Systems) following the manufacturer’s protocol.

Techniques: Derivative Assay, Marker, In Vitro, Expressing, Cell Culture, Co-Culture Assay, Immunofluorescence, Staining, Fluorescence, Knockdown

Journal: Frontiers in Cell and Developmental Biology

Article Title: Integrating scRNA-seq and bulk RNA-seq to explore the differentiation mechanism of human nail stem cells mediated by onychofibroblasts

doi: 10.3389/fcell.2024.1416780

Figure Lengend Snippet: BMP4 induces in vitro differentiation of human NSCs through the TGF-beta signaling pathway. (A) QuSAGE analysis of human nail keratinocyte populations. (B) Effect of recombinant human BMP4 on NSC proliferation. (C) Relative mRNA expression of stem and differentiation marker genes in keratinocytes under different treatments. (D) Western blotting and (E) quantitative analysis of p-SMADs in three groups. The results presented are the mean ± SD of nine biologically independent replicates. (F) Immunofluorescence staining and (G, H) MFI of SMAD4 and p-SMADs in three groups. MFI, Mean Fluorescence Intensity. Scale bars, 100 μm. Unless otherwise stated, the results presented are the mean ± SD of three biologically independent replicates. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Cells were seeded in 6-well plates at a density of 2 × 10 5 cells for 72 h. Then the cell supernatant from different samples was collected and analyzed using a human BMP4 ELISA kit (R&D Systems) following the manufacturer’s protocol.

Techniques: In Vitro, Recombinant, Expressing, Marker, Western Blot, Immunofluorescence, Staining, Fluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Integrating scRNA-seq and bulk RNA-seq to explore the differentiation mechanism of human nail stem cells mediated by onychofibroblasts

doi: 10.3389/fcell.2024.1416780

Figure Lengend Snippet: Proposed model: Onychofibroblast-mediated nail stem cell differentiation links amputation level with digit regeneration capacity. Under homeostatic conditions, BMP4 secreted by onychofibroblasts (OFs) activates the TGF-beta signaling pathway in nail stem cells (NSCs) and induces cell differentiation. It is known that NSCs and the mechanisms governing NSC differentiation are directly related to their ability to orchestrate digit regeneration. In instances of distal amputation, NSCs and OFs remain largely unaffected; regenerating nail epithelial cells can cover the wound site, preserving nail differentiation, and facilitating complete digit regeneration. Conversely, proximal amputation leads to decreased NSCs and OFs, along with diminished differentiation signals, resulting in impaired nail and digit regeneration.

Article Snippet: Cells were seeded in 6-well plates at a density of 2 × 10 5 cells for 72 h. Then the cell supernatant from different samples was collected and analyzed using a human BMP4 ELISA kit (R&D Systems) following the manufacturer’s protocol.

Techniques: Cell Differentiation, Preserving