Stromal inhibition of megakaryocytic induction in HEL cells also correlates with blockade in Rap1 activation.

(A) Coculture of HEL cells with HESS-5 stromal cells blocked the morphologic changes associated with megakaryocytic induction. Morphologic changes are reflected by forward versus right-angle light scatter characteristics on flow cytometry. (B) Coculture of HEL cells with stromal cells blocked surface antigen changes associated with megakaryocytic induction. Shown on the x-axis is mean fluorescent intensity of cells stained with PE-conjugated antibody to GPA. HEL cells either untreated or induced for 48 hours were analyzed by flow cytometry for forward versus side scatter and for glycophorin A expression (GPA). (C) Coculture of HEL cells with stroma blocked Rap1 activation associated with megakaryocytic induction. HEL cells either untreated or induced for 48 hours were analyzed for Rap1 activation as described in “Materials and methods.” In addition, whole cell lysates were immunoblotted for total Rap1 levels.