Journal: International Journal of Molecular Sciences
Article Title: Junctional Adhesion Molecule-C Mediates the Recruitment of Embryonic-Endothelial Progenitor Cells to the Perivascular Niche during Tumor Angiogenesis
doi: 10.3390/ijms21041209
Figure Lengend Snippet: Knockdown of JAM-C reduced in vitro cord-like structures on Matrigel TM . ( A ) The mRNA expression levels of human JAM-C in HUVECs transfected with siRNA against JAM-C or with control siRNA isolated at 24, 48 and 72 h after transfection. The 48 h time point showed the strongest knockdown of JAM-C m-RNA levels. Data are represented by mean ± SD ( n = 3). ( B ) The protein expression level of human JAM-C was analyzed by Western blot in HUVECs transfected with siRNA against JAM-C or with control siRNA, showing a strong decrease in total cellular JAM-C protein levels. Cell surface biotinylation, lysis and JAM-C precipitation were performed 72 h after transfection. Cell lysates from HUVECs transfected with control siRNA or JAM-C siRNA were probed with anti-human JAM-C polyclonal antibody. Equivalent amounts of protein were loaded. As an internal control, anti-human JAM-C was loaded alone. ( C ) The mRNA expression levels of mouse JAM-C showed that JAM-C siRNA treatment decreased JAM-C mRNA levels. ( D ) The protein expression levels of mouse JAM-C showed a strong decrease in total cellular JAM-C protein levels. ( E ) Representative images of cord-like structures from HUVECs untransfected or transfected with control siRNA or with JAM-C siRNA, after 24 h and 48 h on matrigel, are shown. ( F ) Quantitative evaluation of HUVEC cord-like structures, was performed by determining the total tube length. Data are represented by mean ± SD of a representative experiment (*** p < 0.001 compared to the untransfected levels and to control siRNA levels, n = 12). ( G ) Representative images of cord-like structures from e-EPDC untransfected or transfected with control siRNA or with JAM-C siRNA, after 24 h and 48 h on matrigel, are shown. ( H ) Quantitative evaluation of e-EPDC cord-like structures determining the total area of cord structure. Knockdown of JAM-C significantly reduced the cord-like structure when e-EPDCs were transfected with JAM-C siRNA. Data are represented by mean ± SD of a representative experiment; similar results were observed in separate experiments, with each one performed in triplicate (** p < 0.01, *** p < 0.001 compared to untransfected levels and to control siRNA levels, n = 12). Scale bar is 50 μm.
Article Snippet: Cells were double stained for JAM-C and ZOI-1 by incubation for 2 h at room temperature with polyclonal goat anti-mouse JAM-C (R&D, Wiesbaden, Germany) followed by biotinylated anti-goat IgG (Vector Laboratories, Wertheim-Bettingen, Germany), cy2-streptavidin-conjugated antibody (Jackson Immunoresearch Laboratories) and ZO-1 with rabbit anti-mouse ZO-1 (Zymed Lab, San Francisco, CA, USA), followed by goat anti-rabbit IgG cy3-conjugated (Jackson Immunoresearch laboratories).
Techniques: Knockdown, In Vitro, Expressing, Transfection, Control, Isolation, Western Blot, Lysis
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