Effect of cycloheximide and the PKC antagonist RO31-8220 on DAF expression by ECs. HMECs plated at confluence in 35-mm petri dishes (6 × 105 cells/dish) and cultured overnight at 37°C were treated with (A, B) RO31-8220 (1 μmol/L) and (C) cycloheximide (CHX, 1 μg/mL) for 30 minutes before addition of activating factors. The cells were subsequently stimulated with (A, C) TNF- (10 ng/mL) and IFN-γ (500 U/mL), (B) PBu (50 ng/mL), or plain medium for 48 hours. After harvesting, DAF expression was measured by flow cytometry using MoAb 5B2, with MOPC-21 as an isotype-matched negative control. The figure shows the effect of (A, B) specific PKC antagonist RO31-8220 on TNF- and IFN-γ– and PBu-induced DAF expression, respectively, and (C) CHX on TNF- and IFN-γ–induced DAF. CHX also completely inhibited PBu-induced DAF (data not shown). To exclude the effects of nonspecific cytotoxicity, cell viability was assessed by monolayer morphology, cell-counting, and trypan blue exclusion on EC populations before staining (data not shown). To control for any effect of the antagonists on the constitutive expression of DAF, results are expressed as the RFI ratio. The figure is representative of 4 similar experiments.
