An increase in MPL or JAK2 expression, by viral transduction, according to the decreased protein expression, reinduces a proliferation arrest in the presence of TPO. (A) UT7-11oc2-5 clones were transduced with either empty retroviral vector (pMX0) or the vector encoding for MPL (pMX-MPL) and were cultured in the presence of GM-CSF. MPL and p21^CIP1 protein expression were determined by immunoblotting. T+ corresponds to UT7-11oc1 cells exposed to TPO. (B) Cellular proliferation in presence of TPO. Cells were seeded at 20 000 cells/mL (D0) and counted at D5. (C) p21^CIP1 expression was determined for cells exposed to TPO for 3 days, by immunoblotting. (D) SA-β-galactosidase staining in TPO-treated cells at D5. (E) After a 12 h cytokine starvation, UT7-11oc2 cells transduced with either the MPL-expression or control vector were stimulated with TPO. MPL, p21^CIP1 expression and ERK phosphorylation status were then analyzed by immunoblotting. (F) UT7-11oc2-5 clones were transduced with either empty lentiviral vector (TMJ0) or the vector encoding for JAK2 (TMJ-JAK2) and cultured in presence of GM-CSF. MPL and p21^CIP1 protein expressions were determined by immunoblotting. T+ corresponds to UT7-11oc1 cells exposed to TPO. (G) Cellular proliferation in the presence of TPO. Cells were seeded at 20 000 cells/mL and were counted at D7. (H) p21^CIP1 expression was determined by immunoblotting for cells exposed to TPO for 4 days. (I) SA-β-galactosidase staining in TPO-treated cells at D5. (J) After a 12-hour cytokine starvation, UT7-11oc4 cells transduced with either the JAK2-expression or control vector were stimulated with TPO. MPL, p21^CIP1 expression, and ERK phosphorylation status were then analyzed by immunoblotting. Bars: (D,I) 50 μm. Error bars represent standard deviations of 2 independent experiments. NI, noninfected cells.