Figure 4. Phorbol ester-induced differentiation of promonocytic leukemia cells is accompanied by FGF1 export, membrane blebbing, and PS externalization.

Each figure is representative of four independent experiments. (A). PMA treatment induces FGF1 export from U937 cells. U937 cells stably transfected with FGF1 were treated or not treated with 150 nM PMA for 48 h, and then incubated for 110 min at 37°C or 42°C. FGF1 was isolated from conditioned media and detected using immunoblotting. 100% medium and 10% cell lysate were loaded per lane. (B). PMA treatment induces bleb formation in U937 cells. Control and PMA-treated U937 cells were formalin fixed and stained with the Vybrant membrane stain (red). The nuclei were stained with TOPRO3 (blue). Confocal images. Bar – 20 μm. (C). PMA-induced differentiation of U937 cells is accompanied by the externalization of PS. PMA-treated and untreated U937 cells transfected with FGF1:GFP (green) were incubated for 20 min with phycoerythrin-conjugated Annexin V (red), formalin fixed, and examined by confocal microscopy. PS externalization is marked with arrows. Bar - 15.87μm (D,E). Externalization of FGF1 in PMA-treated U937 cells. PMA-treated and untreated U937 cells transfected with FGF1:GFP (green) were incubated for 20min with monoclonal anti-GFP antibodies (red), formalin fixed, incubated with CY3-conjugated secondary antibodies, and examined by confocal microscopy. FGF1 externalization is marked with arrows. Bar – 31.75μm. E: PMA-treated U937 transfected with FGF1:GFP, higher magnification. Processed as in “D.” Additional staining of nuclei with TOPRO-3 (blue). Bar -19μm.