Fig. 2. PMA and TPO induce GPIX surface expression in MK cell lines. A and B, GPIX surface expression in Dami cells was assessed via flow cytometry with an anti-CD42a monoclonal antibody (GRP). Dami cells were growth-arrested overnight and harvested at day 0 (thin lines) and at day 5 (thick lines). The cells were then labeled with anti-CD42a (GPIX) antibody, followed by an FITC-conjugated secondary antibody and examined by flow cytometry. A, cells exposed to PMA (100 nmol/liter) produced a shift to the left after 5 days. B, nontreated cells were used as a negative control. C and D, TPO-inducible expression of GPIX on the surface of UT-7 Epo/Mpl cells was investigated using flow cytometry. Cells were subject to growth arrest overnight then incubated in basal culture medium (Iscove’s modified Dulbecco’s medium/10% FBS/1 unit/ml EPO) supplemented with 20 ng/ml TPO (C) or basal culture medium alone (D). Cells were harvested at day 0 and day 2 and processed as for A and B. Surface expression at day 0 is represented by the thin dotted line and at day 2 by the thick continuous line. GPIX surface expression remained elevated up to 7 days after TPO stimulation (data not shown). The profile of staining using the IgG1 control antibody did not change over the same time period (data not shown). Results are representative of three independent experiments.