TpoR down-modulation by JAK2 V617F requires kinase activity of JAK2. In vivo JAK2 inhibitor treatment restored TpoR levels in mouse and human cells. (A) Flow cytometric analysis (anti-HA) of the indicated Ba/F3 TpoR JAK2 V617F cells incubated with vehicle control, or the indicated inhibitors for 20 hours. Results are derived from the analysis of at least 10 000 events per replicate. Shown are averages of percentages of TpoR expression of 3 independent experiments ± SD, with TpoR levels in the absence of inhibitors being considered as 100%. *Two-tailed (P < .05). **Two-tailed (P < .01). Neither compounds induced toxicity, as assessed by Trypan blue exclusion (not shown). (B) Western blot analysis of TpoR total levels of Ba/F3 TpoR JAK2V617F cells treated or not with the indicated inhibitors for 20 hours. The densitometric ratios between the mature and immature TpoR bands were normalized to that of untreated cells. (C) Confocal analysis of HA-TpoR localization on Ba/F3 TpoR JAK2 V617F cells treated or not with AG490 for 20 hours. (D) Flow cytometric analysis (anti-HA for TpoR cell-surface localization) of the indicated Ba/F3 cell lines. Data are mean ± SD values obtained in 3 flow cytometric measurements, each average being obtained for 10 000 events. Differences between JAK2 V617F and JAK2 V617F K882D cells were statistically significant (P < .01, Student t test). (E) Western blot analysis of paired platelet samples before and after drug treatment from the same KI-JAK2 V617F mouse. Mice were treated either with vehicle (KI-JAK2 V617F-15) or INCB018424 (KI-JAK2 V617F-16, -17, and -18) during 2 weeks (twice a day by gavage). (F) Western blot analysis of platelet extracts from myelofibrosis patients expressing JAK2 V617F (PMF/JAK2V617F-1 and -2). Platelets extracts were obtained before and on ruxolitinib (INCB018424) treatment.