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Fig. 4. PSGL-1-mediated cell aggregation in the presence of versican G3. (A) Two recombinant PSGL-1 constructs, PSGL-1 and PSGL (279-402), were stably expressed in A2058 and U87 cells. Expression of PSGL(1-402) and PSGL(279-402) was assayed by western blotting. Controls were lysates from HL60, U937 and K562 cells. (B) Flow cytometry assays confirmed cell surface localization of these proteins. Blue, negative control; pink, PSGL-1 expression; green, PSGL(279-402) expression. (C) Versican G3-containing medium was incubated with U87 and A2058 cells stably transfected with PSGL(42-402), PSGL(279-402) and a control vector respectively. Ten minutes after cell inoculation, cells expressing PSGL(42-402) or PSGL(279-402) started to aggregate. (D) The G3-containing medium was added to HL60, U937 and K562 cells. Only G3 induced aggregation of HL60 cells (n=3; **P<0.01). (E) G3 was purified through a Ni-NTA affinity column and was analyzed by Coomassie Blue staining and western blotting with 4B6. The medium from vector-transfected cells was used as a control without purification. The strong band in the vector-transfected culture medium stained with Coomassie Blue was BSA. (F) Purified G3 and total proteins from vector-transfected cells were added to HL60 cells and human leukocytes. The purified G3 induced cell aggregation (n=3; **P<0.01).
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