Journal: Biomedicines

Article Title: Soluble JAM-C Ectodomain Serves as the Niche for Adipose-Derived Stromal/Stem Cells

doi: 10.3390/biomedicines9030278

Figure Lengend Snippet: Antibodies.

Article Snippet: To prepare the recombinant protein-coated plates, 1 µg of recombinant JAM-B (rJAM-B; 50464-M08H, SinoBiological, Beijing, China), recombinant JAM-C (rJAM-C; 50465-M08H, SinoBiological, Beijing, China), normal mouse IgG (12-371, Merck Millipore, Burlington, MA, USA) or Cellmatrix type I-A collagen (KP-2020, Nitta gelatin, Osaka, Japan) was placed in 12-well plates overnight at 4 °C.

Techniques:

Journal: Biomedicines

Article Title: Soluble JAM-C Ectodomain Serves as the Niche for Adipose-Derived Stromal/Stem Cells

doi: 10.3390/biomedicines9030278

Figure Lengend Snippet: JAM-B and JAM-C are concentrated on cell membranes of mouse adipose-derived stromal/stem cells (ADSCs). RT-PCR ( A ) and Western blot ( B ) for the indicated molecules in ADSCs derived from three mice (#1–3). Mouse kidney (for Jam-4 ) and spleen tissues (for other junctional adhesion molecules; JAMs) are used as positive controls (Ctrl). P, passage. ( C ) Confocal images of ADSCs stained for the indicated markers. Arrowheads show JAM-B- and JAM-C-immunoreactive signals on the cell membranes of ADSCs. Squares indicate the enlarged areas. Scale bar, 50 µm.

Article Snippet: To prepare the recombinant protein-coated plates, 1 µg of recombinant JAM-B (rJAM-B; 50464-M08H, SinoBiological, Beijing, China), recombinant JAM-C (rJAM-C; 50465-M08H, SinoBiological, Beijing, China), normal mouse IgG (12-371, Merck Millipore, Burlington, MA, USA) or Cellmatrix type I-A collagen (KP-2020, Nitta gelatin, Osaka, Japan) was placed in 12-well plates overnight at 4 °C.

Techniques: Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining

Journal: Biomedicines

Article Title: Soluble JAM-C Ectodomain Serves as the Niche for Adipose-Derived Stromal/Stem Cells

doi: 10.3390/biomedicines9030278

Figure Lengend Snippet: Fluorescence-activated cell sorting (FACS) profiles of the stromal vascular fraction (SVF) of the mouse adipose tissue. ( A ) Association between the expression of mesenchymal stem cell (MSC) markers CD44, CD105, CD140a and Sca-1 and JAM-B or JAM-C expression. ( B ) The JAM-C expression in the Sca1 /CD31 − /CD45 − /Ter119 − cell lineage.

Article Snippet: To prepare the recombinant protein-coated plates, 1 µg of recombinant JAM-B (rJAM-B; 50464-M08H, SinoBiological, Beijing, China), recombinant JAM-C (rJAM-C; 50465-M08H, SinoBiological, Beijing, China), normal mouse IgG (12-371, Merck Millipore, Burlington, MA, USA) or Cellmatrix type I-A collagen (KP-2020, Nitta gelatin, Osaka, Japan) was placed in 12-well plates overnight at 4 °C.

Techniques: Fluorescence, FACS, Expressing

Journal: Biomedicines

Article Title: Soluble JAM-C Ectodomain Serves as the Niche for Adipose-Derived Stromal/Stem Cells

doi: 10.3390/biomedicines9030278

Figure Lengend Snippet: JAM-C is widely distributed in the fat interstitial tissues. ( A – C ) Confocal images of mouse ADSCs stained for the indicated markers. Asterisks indicate lipid droplets in differentiated adipocytes, and arrowheads show colocalization of JAM-C and ether type III collagen or heparan sulfate proteoglycan (HSPG). Squares show the enlarged areas. Scale bars, 50 µm.

Article Snippet: To prepare the recombinant protein-coated plates, 1 µg of recombinant JAM-B (rJAM-B; 50464-M08H, SinoBiological, Beijing, China), recombinant JAM-C (rJAM-C; 50465-M08H, SinoBiological, Beijing, China), normal mouse IgG (12-371, Merck Millipore, Burlington, MA, USA) or Cellmatrix type I-A collagen (KP-2020, Nitta gelatin, Osaka, Japan) was placed in 12-well plates overnight at 4 °C.

Techniques: Staining

Journal: Biomedicines

Article Title: Soluble JAM-C Ectodomain Serves as the Niche for Adipose-Derived Stromal/Stem Cells

doi: 10.3390/biomedicines9030278

Figure Lengend Snippet: The cleaved JAM-C ectodomain is accumulated in the fat interstitial tissues. ( A ) Schematic illustration for detecting full-length JAM-C (fJAM-C) and/or soluble JAM-C (sJAM-C) using JAM-C (N) and JAM-C (C) antibodies (Abs) targeting the N- and C-termini, respectively. ( B ) Knockdown of the Jam3 gene encoding mouse JAM-C in ADSCs using the CRISPR method. ( C ) Western blot for the indicated proteins in the whole-cell lysates and supernatants of the revealed ADSCs. ( D ) Western blot for the indicated proteins in the whole-cell lysates of mouse adult spleen tissue (control), adipose tissue and ADSCs. ( E ) Confocal images of mouse adipose tissue stained with JAM-C (N) and JAM-C (C) Abs. Asterisks indicate lipid droplets in differentiated adipocytes. Squares show the enlarged areas. Scale bar, 50 µm.

Article Snippet: To prepare the recombinant protein-coated plates, 1 µg of recombinant JAM-B (rJAM-B; 50464-M08H, SinoBiological, Beijing, China), recombinant JAM-C (rJAM-C; 50465-M08H, SinoBiological, Beijing, China), normal mouse IgG (12-371, Merck Millipore, Burlington, MA, USA) or Cellmatrix type I-A collagen (KP-2020, Nitta gelatin, Osaka, Japan) was placed in 12-well plates overnight at 4 °C.

Techniques: CRISPR, Western Blot, Staining

Journal: Biomedicines

Article Title: Soluble JAM-C Ectodomain Serves as the Niche for Adipose-Derived Stromal/Stem Cells

doi: 10.3390/biomedicines9030278

Figure Lengend Snippet: Soluble JAM-C functions as the niche for ADSCs. ( A ) Schematic illustration of recombinant JAM-B (rJAM-B) and rJAM-C. S, signal domain; EC, extracellular domain; TM, transmembrane domain; C, cytoplasmic domain. ( B ) Cell adhesion assay for ADSCs grown on culture dishes coated with the indicated proteins. The relative levels are shown in histograms (mean ± SD; n = 5). ( C ) BrdU assay for ADSCs grown on culture plates coated with the indicated proteins. The BrdU/DAPI levels are shown in histograms (mean ± SD; n = 12). Scale bar, 100 µm. ( D ) RT-qPCR for the indicated MSC markers in ADSCs grown on culture dishes coated with the indicated proteins. The relative expression levels are shown in the histograms (mean ± SD; n = 4). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: To prepare the recombinant protein-coated plates, 1 µg of recombinant JAM-B (rJAM-B; 50464-M08H, SinoBiological, Beijing, China), recombinant JAM-C (rJAM-C; 50465-M08H, SinoBiological, Beijing, China), normal mouse IgG (12-371, Merck Millipore, Burlington, MA, USA) or Cellmatrix type I-A collagen (KP-2020, Nitta gelatin, Osaka, Japan) was placed in 12-well plates overnight at 4 °C.

Techniques: Recombinant, Cell Adhesion Assay, BrdU Staining, Quantitative RT-PCR, Expressing

Journal: Biomedicines

Article Title: Soluble JAM-C Ectodomain Serves as the Niche for Adipose-Derived Stromal/Stem Cells

doi: 10.3390/biomedicines9030278

Figure Lengend Snippet: Association between soluble JAM-C and full-length JAM-B on ADSCs. Whole-cell lysates (1% of the input protein samples) and the samples immunoprecipitated (IP) with IgG or JAM-B Ab were immunoblotted (IB) with the indicated Abs. N.S., nonspecific signals.

Article Snippet: To prepare the recombinant protein-coated plates, 1 µg of recombinant JAM-B (rJAM-B; 50464-M08H, SinoBiological, Beijing, China), recombinant JAM-C (rJAM-C; 50465-M08H, SinoBiological, Beijing, China), normal mouse IgG (12-371, Merck Millipore, Burlington, MA, USA) or Cellmatrix type I-A collagen (KP-2020, Nitta gelatin, Osaka, Japan) was placed in 12-well plates overnight at 4 °C.

Techniques: Immunoprecipitation

Journal: Biomedicines

Article Title: Soluble JAM-C Ectodomain Serves as the Niche for Adipose-Derived Stromal/Stem Cells

doi: 10.3390/biomedicines9030278

Figure Lengend Snippet: A Soluble JAM-C couples with JAM-B to promote the ADSC adhesion and niche function. ( A ) Knockdown of the Jam2 gene encoding mouse JAM-B in ADSCs using the CRISPR method. ( B ) Western blot for the indicated proteins in the whole-cell lysates of the revealed ADSCs. ( C ) Cell adhesion assay for ADSCs grown in the indicated culture conditions. The relative levels are shown in histograms (mean ± SD; n = 8). ( D ) RT-qPCR for the indicated MSC markers in ADSCs cultivated in the indicated culture conditions. The relative expression levels are shown in the histograms (mean ± SD; n = 4). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: To prepare the recombinant protein-coated plates, 1 µg of recombinant JAM-B (rJAM-B; 50464-M08H, SinoBiological, Beijing, China), recombinant JAM-C (rJAM-C; 50465-M08H, SinoBiological, Beijing, China), normal mouse IgG (12-371, Merck Millipore, Burlington, MA, USA) or Cellmatrix type I-A collagen (KP-2020, Nitta gelatin, Osaka, Japan) was placed in 12-well plates overnight at 4 °C.

Techniques: CRISPR, Western Blot, Cell Adhesion Assay, Quantitative RT-PCR, Expressing

Journal: Biomedicines

Article Title: Soluble JAM-C Ectodomain Serves as the Niche for Adipose-Derived Stromal/Stem Cells

doi: 10.3390/biomedicines9030278

Figure Lengend Snippet: Schematic model for the regulation of ADSC functions by soluble JAM-C. The soluble JAM-C (sJAM-C) deposited on the extracellular matrix interacts with JAM-B and partly with JAM-C on ADSC. sJAM-C/JAM-B signaling reaches the nucleus (dashed arrow) and stimulates cellular proliferation and expression of MSC markers (arrows). MSC, mesenchymal stromal/stem cell.

Article Snippet: To prepare the recombinant protein-coated plates, 1 µg of recombinant JAM-B (rJAM-B; 50464-M08H, SinoBiological, Beijing, China), recombinant JAM-C (rJAM-C; 50465-M08H, SinoBiological, Beijing, China), normal mouse IgG (12-371, Merck Millipore, Burlington, MA, USA) or Cellmatrix type I-A collagen (KP-2020, Nitta gelatin, Osaka, Japan) was placed in 12-well plates overnight at 4 °C.

Techniques: Expressing