Figure 1.
Figure 1. Stimulation of mono-DCs through hOSCAR promotes cell survival in the absence of survival factors by a PI3K-, ERK-dependent pathway. Mono-DCs were washed 4 times to remove GM-CSF and IL-4, before stimulation of the cells with plastic-coated mAb (MOPC21, anti-CD13, anti-hOSCAR), 200 ng/mL GM-CSF, or 20 ng/mL TNF. Data shown are representative of 3 independent experiments. (A) Mono-DCs were stimulated as described and photographed after 3 days. Cells were visualized using an inverted Olympus CK40 microscope (Olympus, Tokyo, Japan) with a 20×/0.50 aperture objective; final total magnification, × 200. Images were captured with a Nikon camera (Nikon, Melville, NY) and imported into Adobe Photoshop (Adobe Systems, San Jose, CA). (B) After stimulation for the indicated time, cells were harvested and counted by exclusion of dead cells with blue trypan. Cell recovery is expressed as percentage of cells put into culture at day 0. □ indicates medium only; ▪, MOPC21; ○, anti-hOSCAR; and ⬡, GM-CSF. Survival curve displays the mean and standard deviation of 3 independent cell counts from 1 representative experiment. (C) After 3 days of stimulation as described, cells were analyzed for annexin V binding, DiOC6(3) incorporation, and intracellular staining by anti–Bcl-2 and Bcl-x. Numbers in the corners correspond to the percentage of positive cells for annexin V and DiOC6(3) analysis, and indicate specific mean fluorescence intensity for Bcl-2 and Bcl-x staining (shaded histogram). The dotted line shows the binding of an isotype control mAb to the cells. (D) Lysates of mono-DCs stimulated for 3 days, as indicated, were analyzed for expression of Bcl-x isoforms by Western blot. The antiapoptotic long isoform of BcL-x corresponds to the band of 28 kDa. (E) Mono-DCs were stimulated for 3 days, as described, in the presence of PI3K inhibitor (LY294002) or ERK-pathway inhibitor (PD98059). The percentage of apoptotic cells and expression of Bcl-2 were analyzed by annexin V–FITC binding and anti–Bcl-2 labeling. The data were expressed as percentages for annexin V–FITC and ΔMFI (mean fluorescence intensity minus fluorescence detected with isotype control). □ indicates medium only; ▪, anti-hOSCAR; and ▦, GM-CSF.

Stimulation of mono-DCs through hOSCAR promotes cell survival in the absence of survival factors by a PI3K-, ERK-dependent pathway. Mono-DCs were washed 4 times to remove GM-CSF and IL-4, before stimulation of the cells with plastic-coated mAb (MOPC21, anti-CD13, anti-hOSCAR), 200 ng/mL GM-CSF, or 20 ng/mL TNF. Data shown are representative of 3 independent experiments. (A) Mono-DCs were stimulated as described and photographed after 3 days. Cells were visualized using an inverted Olympus CK40 microscope (Olympus, Tokyo, Japan) with a 20×/0.50 aperture objective; final total magnification, × 200. Images were captured with a Nikon camera (Nikon, Melville, NY) and imported into Adobe Photoshop (Adobe Systems, San Jose, CA). (B) After stimulation for the indicated time, cells were harvested and counted by exclusion of dead cells with blue trypan. Cell recovery is expressed as percentage of cells put into culture at day 0. □ indicates medium only; ▪, MOPC21; ○, anti-hOSCAR; and ⬡, GM-CSF. Survival curve displays the mean and standard deviation of 3 independent cell counts from 1 representative experiment. (C) After 3 days of stimulation as described, cells were analyzed for annexin V binding, DiOC6(3) incorporation, and intracellular staining by anti–Bcl-2 and Bcl-x. Numbers in the corners correspond to the percentage of positive cells for annexin V and DiOC6(3) analysis, and indicate specific mean fluorescence intensity for Bcl-2 and Bcl-x staining (shaded histogram). The dotted line shows the binding of an isotype control mAb to the cells. (D) Lysates of mono-DCs stimulated for 3 days, as indicated, were analyzed for expression of Bcl-x isoforms by Western blot. The antiapoptotic long isoform of BcL-x corresponds to the band of 28 kDa. (E) Mono-DCs were stimulated for 3 days, as described, in the presence of PI3K inhibitor (LY294002) or ERK-pathway inhibitor (PD98059). The percentage of apoptotic cells and expression of Bcl-2 were analyzed by annexin V–FITC binding and anti–Bcl-2 labeling. The data were expressed as percentages for annexin V–FITC and ΔMFI (mean fluorescence intensity minus fluorescence detected with isotype control). □ indicates medium only; ▪, anti-hOSCAR; and ▦, GM-CSF.

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