Figure 5
Figure 5. Function of DEPTOR in leukocyte-endothelial interactions in vitro. (A-C) Confluent cultures of untransfected and control siRNA- and DEPTOR siRNA-transfected HUVECs were cocultured with freshly isolated human PBMCs at 37°C for 10 to 60 minutes. Subsequently, the cultures were washed 3 times, and the number of adherent PBMCs were evaluated by microscopy or FACS analysis. (A) Representative photomicrographs of HUVECs transfected with control or DEPTOR siRNAs and cultured with human PBMCs for 10, 30, and 60 minutes. Cells cultured with TNFα-treated ECs (100 U/mL for 6 hours) are illustrated as a positive control. Microscopy was carried out (10× objective) using a digital inverted microscope (AMG Evos XL Core; Fisher Scientific). (B) Representative FACS dot plots of the patterns of forward scatter (FSC) and side scatter (SSC) for 10 000 cells per group, illustrating the percentage of adherent PBMCs to each group of HUVECs after 60 minutes. The expression of CD45 within the PBMC gate (lower left panel, open histogram), as well as CD45 and CD31 within the HUVEC gate (lower center and right panels, open histograms) are shown vs isotype antibody as a control (shaded histogram). The bar graph illustrates the mean adhesion index of PBMC (±SEM) to ECs from 5 independent experiments. (C) Representative FACS plots of CD3+ T cells within each PBMC gate shown in B. The bar graph shows the mean adhesion index of CD3+ T cells (±SEM) to ECs from 5 independent experiments. (D-E) PBMCs and freshly isolated CD3+ T cells were labeled with CFSE (5 μM) prior to coculture in adhesion assays with untransfected, control siRNA, or 2 DEPTOR siRNA-transfected HUVECs. ECs were cultured in the absence or presence of rapamycin (labeled R, 10 ng/mL) and/or U0126 (labeled U, 10 μM) for 24 hours prior to each assay. After 60 minutes of coculture, nonadherent cells were removed by washing, and adherent leukocytes were evaluated by measurement of fluorescence in each well. The number of adherent leukocytes was calculated based on a standard curve, as described in Materials and methods. The bar graphs show the mean number (±SEM) of adherent (D) PBMCs and (E) CD3+ T cells from 5 independent experiments. Data from ECs transfected with DEPTOR siRNA1 #1 or #2 were pooled for analysis in the bar graphs. *P < .05, **P < .005, ***P < .001.

Function of DEPTOR in leukocyte-endothelial interactions in vitro. (A-C) Confluent cultures of untransfected and control siRNA- and DEPTOR siRNA-transfected HUVECs were cocultured with freshly isolated human PBMCs at 37°C for 10 to 60 minutes. Subsequently, the cultures were washed 3 times, and the number of adherent PBMCs were evaluated by microscopy or FACS analysis. (A) Representative photomicrographs of HUVECs transfected with control or DEPTOR siRNAs and cultured with human PBMCs for 10, 30, and 60 minutes. Cells cultured with TNFα-treated ECs (100 U/mL for 6 hours) are illustrated as a positive control. Microscopy was carried out (10× objective) using a digital inverted microscope (AMG Evos XL Core; Fisher Scientific). (B) Representative FACS dot plots of the patterns of forward scatter (FSC) and side scatter (SSC) for 10 000 cells per group, illustrating the percentage of adherent PBMCs to each group of HUVECs after 60 minutes. The expression of CD45 within the PBMC gate (lower left panel, open histogram), as well as CD45 and CD31 within the HUVEC gate (lower center and right panels, open histograms) are shown vs isotype antibody as a control (shaded histogram). The bar graph illustrates the mean adhesion index of PBMC (±SEM) to ECs from 5 independent experiments. (C) Representative FACS plots of CD3+ T cells within each PBMC gate shown in B. The bar graph shows the mean adhesion index of CD3+ T cells (±SEM) to ECs from 5 independent experiments. (D-E) PBMCs and freshly isolated CD3+ T cells were labeled with CFSE (5 μM) prior to coculture in adhesion assays with untransfected, control siRNA, or 2 DEPTOR siRNA-transfected HUVECs. ECs were cultured in the absence or presence of rapamycin (labeled R, 10 ng/mL) and/or U0126 (labeled U, 10 μM) for 24 hours prior to each assay. After 60 minutes of coculture, nonadherent cells were removed by washing, and adherent leukocytes were evaluated by measurement of fluorescence in each well. The number of adherent leukocytes was calculated based on a standard curve, as described in Materials and methods. The bar graphs show the mean number (±SEM) of adherent (D) PBMCs and (E) CD3+ T cells from 5 independent experiments. Data from ECs transfected with DEPTOR siRNA1 #1 or #2 were pooled for analysis in the bar graphs. *P < .05, **P < .005, ***P < .001.

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