Journal: eLife

Article Title: Integrin alpha11 is an Osteolectin receptor and is required for the maintenance of adult skeletal bone mass

doi: 10.7554/eLife.42274

Figure Lengend Snippet: ( A, B ) The human ( A ) and mouse ( B ) Osteolectin proteins contain RGD and LDT sequences. ( C ) Alignment of Osteolectin amino acid sequences shows that the RGD and LDT domains are evolutionarily conserved among bony vertebrates. ( D, E ) RNA-seq analysis of integrin α ( D ) and β ( E ) subunits in PDGFRα + CD45 - Ter119 - CD31 - bone marrow stromal cells from enzymatically dissociated adult bone marrow (n = 2 independent samples). These cells are uniformly positive for LepR expression . ( F ) RNA-seq analysis of Itga1 , Itga6 , Itga11 , and Itgav in PDGFRα + CD45 - Ter119 - CD31 - bone marrow stromal cells, VE-Cadherin + bone marrow endothelial cells, and whole bone marrow cells (n = 2 independent samples per cell population). ( G ) Itga11 expression in cell populations from mouse bone marrow by qRT-PCR (n = 3 independent samples per cell population). The markers used for the isolation of each cell population are shown in . ( H ) In MC3T3-E1 preosteoblast cells expressing Flag-tagged Osteolectin, anti-Flag antibody co-immunoprecipitated endogenous integrin β1 and integrin α11 with Flag-tagged Osteolectin (results are representative of two independent experiments). ( I ) Recombinant human Osteolectin (rhOln) selectively bound to recombinant human integrin α 11 β 1 and α 10 β 1 , but not to other integrins (n = 3 independent experiments). ( J ) Integrin α11β1 bound Osteolectin and recombinant human Pro-Collagen 1α (rhCol1A) with similar affinities, but not bovine serum albumin (BSA) (n = 3 independent experiments). ( K ) Osteolectin, but not Pro-Collagen 1α, promoted osteogenic differentiation by MC3T3-E1 cells and human bone marrow stromal cells (n = 3 independent experiments). ( L ) 200 nM RGDS peptide inhibited the binding of integrin α11β1 to recombinant human Osteolectin. ( M ) 100 μM RGDS peptide inhibited osteogenic differentiation by MC3T3-E1 cells and human bone marrow stromal cells in response to 30 ng/ml of recombinant human Osteolectin. All numerical data reflect mean ±standard deviation. Statistical significance was determined with one-way ( G ) or two-way ANOVAs with Dunnett’s multiple comparisons tests ( K ) or Tukey’s multiple comparisons tests ( M ). 10.7554/eLife.42274.004 Figure 1—source data 1. Data for .

Article Snippet: Peptide, recombinant protein , recombiant Integrin α4β1 protein , R and D Systems , 5668-A4 , .

Techniques: RNA Sequencing, Expressing, Quantitative RT-PCR, Isolation, Immunoprecipitation, Recombinant, Binding Assay, Standard Deviation

Journal: eLife

Article Title: Integrin alpha11 is an Osteolectin receptor and is required for the maintenance of adult skeletal bone mass

doi: 10.7554/eLife.42274

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , recombiant Integrin α4β1 protein , R and D Systems , 5668-A4 , .

Techniques: Recombinant, Diagnostic Assay, Cell Culture, Protease Inhibitor, Western Blot, Reverse Transcription, Enzyme-linked Immunosorbent Assay, Fractionation

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Talin2 mediates secretion and trafficking of matrix metallopeptidase 9 during invadopodium formation.

doi: 10.1016/j.bbamcr.2020.118693

Figure Lengend Snippet: Fig. 2. Ablation of talin2 inhibited MMP2 and MMP9 secretion and caused a reduction in MMP9 vesicles targeting to ventral plasma membrane. A. Secreted MMP2 and MMP9 in talin2-KO MDA-MB-231 cells and CRISPR control cells were collected from serum-free supernatants, concentrated and determined by Western blotting. Data are presented as mean ± SEM of 3 independent experiments. t-test, *P < 0.05, ***P < 0.01. B. MMP2 and MMP9 mRNA levels in talin2-KO MDA-MB-231 cells and CRISPR control cells were measured using real-time qPCR. Data are presented as mean ± SEM of 5 independent experiments. t-test, *P < 0.05. C–F, Talin2-KO MDA-MB-231 cells and CRISPR control cells were plated on gelatin for 36 h with 1% FBS and 0.05 μg/ml HGF. C. Cells were co-stained with anti-β1- integrin and anti-MMP9 antibodies. Representative TIRF and brightfield images are shown. D. Intensities of all vesicles from all imaged cells were measured as their maximum intensity, and presented in form of a histogram normalized for number of cells (Control, n = 38; TLN2-KO#1, n = 44; TLN2-KO#2, n = 59), the peak at the maximum is connected with super-bright vesicles beyond dynamic range of the microscope camera. Data are representative of three independent experiments. E. The same data was presented using box-whisker plot. Data presented as 10-25-50-75-90 percentiles. U test, ***P < 0.001. F. Total intensity of MMP9 fluorescence per cell, measured as a sum of intensity maxima of MMP9 vesicles. Data presented as mean value ± SEM. U test, *P < 0.05, ***P < 0.001.

Article Snippet: Anti-β1-integrin mouse monoclonal antibody (clone P5D2) was from R&D Systems.

Techniques: Clinical Proteomics, Membrane, CRISPR, Control, Western Blot, Staining, Microscopy, Whisker Assay, Fluorescence

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Talin2 mediates secretion and trafficking of matrix metallopeptidase 9 during invadopodium formation.

doi: 10.1016/j.bbamcr.2020.118693

Figure Lengend Snippet: Fig. 7. Scheme of talin2-mediated MMP9 trafficking pathway. MMP9 secretory vesicles (1) released from Golgi apparatus are transported towards ventral membrane where complex of talin2, β1-integrin and unknown factor (either cytoplasmic like moesin-NHE-1 or calpain, or membrane like PIP2) mediate their docking (A) and secretion (B), consequently. In case of absence of the complex, MMP9 vesicles are trafficked towards degradation process (C) in ly- sosomes (6) through early and late endosomes (2 and 3, respectively), with possible merging with autophagosomes (4) to create amphisome (5).

Article Snippet: Anti-β1-integrin mouse monoclonal antibody (clone P5D2) was from R&D Systems.

Techniques: Membrane