MCs efficiently adhered to OP9-Dll1, -Dll4, -Jag1, and -Jag2, but not OP9-Dll3, than to OP9-Ctrl.

<p>(A and B) Flow cytometric analysis of the expression of (A) Dll1, Dll3, Dll4, Jag1, and Jag2 on OP9 stromal cells transduced with each Notch ligand gene, and (B) Notch receptors on MCs after staining with specific mAbs (open histograms) or isotype-matched control mAbs (filled histograms). (C) Total RNA was analyzed by RT-PCR for the expression of Notch receptors in MCs and OP9-Ctrl cells. (D) Relative expression levels of <i>Notch1</i> and <i>Notch2</i> to <i>Gapdh</i> in MCs were analyzed by quantitative RT-PCR. Data represent the mean ± SEM of 3 independent experiments. (E and F) An adhesion assay for MCs on each OP9 cell (E) in a 48-well plate for 60 min and (F) in 96-well plates with serial incubation times of 5, 15, 30, 60, and 120 min. Data represent the percentages of non-adherent MCs (mean ± SEM of triplicate cultures) (*p<0.05 significantly different from OP9-Ctrl at each time point, the Student’s <i>t-</i>test).</p

Enhanced adhesion of MCs by Notch ligands involved both Notch1 and Notch2 on MCs.

<p>An adhesion assay (60 min) for MCs on each OP9 cell in a 96-well plate (A and B) with or without 10 µg/ml of the indicated polyclonal Ab (pAb) and (C) with control pAb (20 µg/ml), anti-Notch2 pAb (10 µg/ml) plus control pAb (10 µg/ml) or anti-Notch2 pAb (10 µg/ml) plus anti-Notch1 pAb (10 µg/ml). Data represent the percentages of non-adherent MCs (mean ± SEM of triplicate cultures) (*p<0.05, the Student’s <i>t-</i>test). Cultures with pAbs contained (A and B) 1.0% PBS (vol/vol) and 38.5 µM NaN₃ and (C) 2.0% PBS (vol/vol) and 76.9 µM NaN₃, which had no effect on the adhesion of MCs.</p

Notch signaling in stromal cells or MCs did not account for the enhanced adhesion.

<p>(A) An adipocyte differentiation assay of OP9-Ctrl cells stimulated with immobilized Notch ligands or human IgG1 (control) for 5 days in the presence of DAPT (10 µM) or the same volume of DMSO (control, 0.1% vol/vol). Data represent the numbers of adipocytes in a field (magnification; x200) in the center of the wells (mean ± SEM of triplicate cultures) (*p<0.05 significantly different from IgG1, the Student’s <i>t-</i>test). ND: not detected. (B) OP9-Ctrl cells were stimulated with each immobilized Notch ligand or human IgG1 (control) for 2 days in a 48-well plate, and an adhesion assay (60 min) for MCs was then performed. (C) An adhesion assay (60 min) for MCs on each OP9 cell with DAPT (10 µM) or the same volume of DMSO (control, 0.1% vol/vol). (B and C) Data represent the percentages of non-adherent MCs (mean ± SEM of triplicate cultures). (B) *p<0.05 significantly different from IgG1 and (C) no significant differences between the responses with DMSO and DAPT on the same OP9 cells (the Student’s <i>t-</i>test).</p

Reduction in Notch2 on MCs did not have marked effects on the enhanced adhesion.

<p>MCs were analyzed 48 hours after transfection with siRNA against Notch2 or control siRNA. (A) Relative expression levels of <i>Notch1</i> and <i>Notch2</i> to <i>Gapdh</i> in MCs transfected with each siRNA were analyzed by quantitative RT-PCR. Data represent the mean ± SEM of three independent experiments. (*p<0.05 significantly different from the control siRNA treatment, the Student’s <i>t-</i>test) (B) Flow cytometric analysis of the expression of Notch2 and Kit on MCs transfected with each siRNA after staining with specific mAbs (open histograms) or isotype-matched control mAbs (filled histograms). Representative histograms from one of three independent experiments are shown. Numbers indicate the relative mean fluorescence intensities (MFIs) of specific mAbs relative to that of the control siRNA treatment (MFIs of specific mAbs were normalized by the MFIs of control mAbs) (mean ± SEM of three independent experiments). (C) An adhesion assay (60 min) for MCs transfected with each siRNA on each OP9 cell in a 96-well plate. Data represent the percentages of non-adherent MCs (mean ± SEM of triplicate cultures) (*p<0.05, the Student’s <i>t-</i>test).</p