JAK2 shRNA expression in UT7-11oc1 cells restores cellular proliferation and inhibits the megakaryocytic phenotype in the presence of TPO. (A) shRNA lentiviral transductions of UT7-11oc1 cells were efficient to inhibit JAK2 mRNA expression. UT7-11oc1 cells were infected with shJAK2-1-4, cultured in the presence of GM-CSF and JAK2 mRNA expression (relative to HPRT) evaluated by quantitative real-time PCR. (B) shRNA lentiviral transductions of UT7-11oc1 cells were efficient to inhibit JAK2 protein expression. UT7-11oc1 cells were infected with shJAK2-1-4, cultured in the presence of GM-CSF and JAK2 protein expression (relative to actin) evaluated by immunoblotting. (C) Parallel to JAK2 expression, total and cell-surface MPL expression was determined by immunoblotting. The shRNA-expressing cells were seeded at 25 000 cells/mL and were studied at D4. The shJAK2-1-4-expressing cells were characterized by a cellular proliferation (D), the lack of p21^CIP1 protein expression (E), morphologic changes and SA-β-galactosidase staining (F), and by a decreased megakaryocytic markers (CD41) expression (G) in the presence of TPO. Bars: (E) 50 μm. Error bars represent mean ± standard deviation of 2 independent experiments.