Figure 6
TpoR induces an antiproliferative effect at high ligand concentration. (A) Proliferation assays of the indicated Ba/F3 cells as a function of the concentration of ligand Tpo. Ba/F3 TpoR cells responded to Tpo by proliferation, whereas Ba/F3 TpoR JAK2 cells responded by a stop in cell proliferation at concentrations of Tpo more than 0.5 ng/mL. Ba/F3 TpoR TYK2 cells stopped proliferation at Tpo concentrations more than 10 ng/mL. Cell proliferation was measured by Cell TiterGlo assays 48 hours after seeding. (B) HA-TpoR total levels of Ba/F3 TpoR JAK2wt cells in the indicated conditions were analyzed by Western blot of NP40 buffer extracts using anti-JAK2, anti-HA, and anti–β-actin antibodies. (C) Left panel: The indicated cell lines were obtained by retroviral infection and cell sorting and amplification for a short time, so that down-regulation of TpoR by JAK2 V617F to levels below those of Ba/F3 TpoR cells did not yet occur. Proliferation assays indicate that addition of 200 ng/mL Tpo led to proliferation in Ba/F3 TpoR cells and to a stop in proliferation in cells overexpressing JAK2 or JAK2 V617F and TpoR. Right panel: Proliferation assays performed on the indicated cell lines after longtime amplification so that TpoR down-regulation occurs in presence of JAK2 V617F. Data are mean of triplicates ± SD. Differences between TpoR columns in the +Tpo (200 ng/mL) and all other conditions (TpoR JAK2 and TpoR JAK2 V617F) are statistically significant at P < .05 (Student t test both on the left and right panels). (D) The location of the 3 main cytosolic tyrosine residues in TpoR cytosolic domain. (E) The indicated Ba/F3 cell lines coexpressing either JAK2 or JAK2 V617F and TpoR variants carrying tyrosine to phenylalanine mutations were analyzed for Tpo (200 ng/mL)-induced proliferation immediately after retroviral transduction, before down-modulation of TpoR occurred in JAK2 V617F cells. (A,C-E) Averages of 3 replicates ± SD of one representative experiment of at least 2 independent experiments. Differences between TpoR and TpoR JAK2 or TpoR JAK2 V617F are statistically significant at P < .05 (Student t test for the Y626F mutation), and also between all Y592F and Y626F mutants, as well as between TpoR JAK2 V617F and TpoR Y631F JAK2 V617F cells.

TpoR induces an antiproliferative effect at high ligand concentration. (A) Proliferation assays of the indicated Ba/F3 cells as a function of the concentration of ligand Tpo. Ba/F3 TpoR cells responded to Tpo by proliferation, whereas Ba/F3 TpoR JAK2 cells responded by a stop in cell proliferation at concentrations of Tpo more than 0.5 ng/mL. Ba/F3 TpoR TYK2 cells stopped proliferation at Tpo concentrations more than 10 ng/mL. Cell proliferation was measured by Cell TiterGlo assays 48 hours after seeding. (B) HA-TpoR total levels of Ba/F3 TpoR JAK2wt cells in the indicated conditions were analyzed by Western blot of NP40 buffer extracts using anti-JAK2, anti-HA, and anti–β-actin antibodies. (C) Left panel: The indicated cell lines were obtained by retroviral infection and cell sorting and amplification for a short time, so that down-regulation of TpoR by JAK2 V617F to levels below those of Ba/F3 TpoR cells did not yet occur. Proliferation assays indicate that addition of 200 ng/mL Tpo led to proliferation in Ba/F3 TpoR cells and to a stop in proliferation in cells overexpressing JAK2 or JAK2 V617F and TpoR. Right panel: Proliferation assays performed on the indicated cell lines after longtime amplification so that TpoR down-regulation occurs in presence of JAK2 V617F. Data are mean of triplicates ± SD. Differences between TpoR columns in the +Tpo (200 ng/mL) and all other conditions (TpoR JAK2 and TpoR JAK2 V617F) are statistically significant at P < .05 (Student t test both on the left and right panels). (D) The location of the 3 main cytosolic tyrosine residues in TpoR cytosolic domain. (E) The indicated Ba/F3 cell lines coexpressing either JAK2 or JAK2 V617F and TpoR variants carrying tyrosine to phenylalanine mutations were analyzed for Tpo (200 ng/mL)-induced proliferation immediately after retroviral transduction, before down-modulation of TpoR occurred in JAK2 V617F cells. (A,C-E) Averages of 3 replicates ± SD of one representative experiment of at least 2 independent experiments. Differences between TpoR and TpoR JAK2 or TpoR JAK2 V617F are statistically significant at P < .05 (Student t test for the Y626F mutation), and also between all Y592F and Y626F mutants, as well as between TpoR JAK2 V617F and TpoR Y631F JAK2 V617F cells.

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