UT7-11oc2-7: clones derived from UT7-MPL (UT7-11oc1) cells escaping TPO-induced cellular proliferation arrest. Characterization of UT7-11oc2-7 clones according to MPL/JAK2 protein expression. We derived clones named UT7-11oc2-7 by culturing UT7-11oc1 cells in the presence of TPO. (A) Resurgence of cellular proliferation after a midterm (D10-D14) culture of UT7-11oc1 cells with TPO. Cells escaping TPO-induced proliferation arrest were named UT7-11oc2 (first clone derived). Viable cells were counted using Trypan blue exclusion. (B) UT7-11oc2 cells were cultured in the presence of either GM-CSF or TPO, and cell proliferation was compared with that of UT7-11oc1 cells exposed to TPO. (C) May Grünwald Giemsa staining in TPO-treated cells. (D) CD41 expression measured by flow cytometry in TPO-treated cells. (E) Differences in cell signaling, notably activation of MAPK pathway, and p21CIP1 expression for UT7-11oc1 and UT7-11oc2 cells. After a 12-hour cytokine starvation, UT7-11oc1 and UT7-11oc2 cells were restimulated by TPO during the indicated times, and cell signaling was studied by immunoblotting. (F) Clusterization of UT7-11oc2-7 clones according to TPO-induced gene expression profile. After a 12-hour cytokine starvation, UT7-11oc1 and UT7-11oc2-7 cells were stimulated with TPO for 24 hours. TPO-induced gene expression for each derived clone (UT7-11oc2-7)—relative to that of UT7-11oc1 cells—was determined by microarray analysis, and gene expression profiles obtained for the different UT7-11oc2-7 clones were compared with each other. (G) UT7-11oc1 cells and UT7-11oc2-7 clones were cultured with GM-CSF and MPL and JAK2 protein expression analyzed by immunoblotting. (H) MPL cell-surface expression was analyzed by flow cytometry for cells cultured in the presence of GM-CSF. Mean fluorescence intensity was 1190 for UT7-11oc1 cells vs 743 for UT7-11oc2 cells. Bars: (C) 50 μm. Error bars represent mean ± standard deviation of 3 independent experiments.
