CFU-Mk generation from CD34⁺cells of CALR- and JAK2V617F-mutated patients is dependent on IL-6. Immunomagnetically isolated CD34⁺ cells from healthy controls (HC; n = 6), CALR-mutated (n = 10), or JAK2V617F-mutated (n = 7) patients were seeded in MegaCult medium depleted of IL-6; CFU-Mk colonies were counted on day 12. Data are expressed as percent of control cultures in media containing IL-6 (A). The levels of IL-6 mRNA were assessed by qRT-PCR in CD34⁺ cells from CALR- (n = 8) and JAK2V617F-mutated (n = 8) MPN patients and healthy controls (HC; n = 7). Data are expressed as relative quantity (RQ) to reference RNAseP mRNA. (B) The symbol ° points to 3 CALR type 2–mutated patients, whereas the remaining 5 were typical DEL mutation. (C) The expression of p-STAT3 was evaluated by confocal microscopy in isolated CD34⁺ cells from CALR- or JAK2V617F-mutated patients or healthy subjects (n = 3 for each category; ×200 magnification). Random chosen cells from 1 healthy control, CALR- or JAK2V617F-mutated patient are presented. DAPI (4′,6-diamidino-2-phenylindole) dye was used to identify the nucleus. (D-G) The expression of gp130 and IL-6R on the cell membrane of CALR^mut, JAK2V617F^mut patients and healthy controls was assessed by flow cytometry; the percentage of positive cells (D-E) and the mean fluorescent intensity (F-G) are shown. (H-J) Dose-dependent inhibition of CFU-Mk generation in clonogenic cultures established from CALR-mutated (n = 6) or JAK2V617F-mutated (n = 5) patients and healthy controls (HC; n = 6). SC144, TCZ, and an anti–IL-6 antibody were added at the time of culture initiation. CFU-Mk colonies from triplicate dishes were counted after 12 days, and the number of colonies was expressed as percent of controls in the absence of respective drugs. Respective IC50 values (mean plus standard error [SE]) are detailed in panel K. All P values were determined by Student t test (*P < .05; **P < .01; ***P < .001).