RUX maintains murine and human MPN HSPCs. (A) Schematic diagram showing the in vitro functional assays of murine ESLAM HSCs (CD45⁺CD150⁺CD48⁻EPCR⁺) treated with RUX or DMSO. ESLAM HSCs were FACS isolated from CALRdel/del (n = 4) mutant mice and were then cultured for 7 days in IL-3/IL-6/SCF media⁶⁷ with DMSO or 250 nM of RUX before analysis by flow cytometry. (B) Bar plots showing cell number per well in HSC-derived cultures treated with vehicle or 250 nM RUX after 7 days (mean ± SEM). (C) Bar plots showing the proportion of cells that expressed lineage-positive markers (Ter119⁺/Ly6g⁺/CD11b⁺/B220⁺/CD3e⁺) after 7 days in culture with DMSO or 250 nM RUX (mean ± SEM). Asterisks indicate significant differences as determined by Student t tests (∗∗∗P < .001; ∗∗P < .01; ∗P < .05). (D) Schematic diagram showing the serial replating assays that investigated the effect of RUX on ESLAM HSCs isolated from WT and CALRdel/del mutant mice. Sorted ESLAM HSCs were cultured for 7 days in IL-3/IL-6/SCF media⁶⁷ with DMSO or 250 nM RUX and then subjected to serial colony replating assays. (E) Bar plots showing the fold change in the number of colonies produced by HSC-derived cultures treated with vehicle or 250 nM RUX for 7 days, normalized to the number of colonies produced by vehicle-treated cultures at each week of replating. The results are from 2 independent experiments and are shown as mean ± SEM. Asterisks indicate significant differences as determined by Mann-Whitney U tests (∗P < .05) (F) Schematic diagram showing that HSCs (MPP1–LT-HSCs; CD34⁺CD38⁻CD45RA⁻) cells were sorted from healthy human platelet apheresis donor cone samples or from the peripheral blood of patients with myelofibrosis into 96-well plates (400 cells per well) and cultured in high-cytokine, serum-free medium (EXPER cytokine media)⁷² with scaled doses of RUX or vehicle control (DMSO). After 7 days, the HSC-derived cultures were plated in serial colony replating assays in methylcellulose. Healthy donors were all male and between 48 and 69 years of age. Among the donor patients with myelofibrosis, 3 patients carried a JAK2 V617F mutation and were all male between the ages of 65 to 70 years and 1 donor carried a CALR 52 bp deletion mutation and was a 70 year old female at the time of sample collection. (G) Bar plots showing the fold change in the number of colonies produced by HSPCs that were isolated from healthy donors and cultured for 7 days in the presence of RUX, normalized to the number of colonies that were produced by HSPCs after culturing for 7 days with DMSO. The data are shown as log₂(fold change) from DMSO. Left showing fold change in colony numbers in the first round of colony formation (2 weeks in methylcellulose). Right showing fold change in colony numbers in the second round of colony formation (4 weeks in methylcellulose). From the data of the 4 healthy donors, each dot represents the mean fold change between technical replicates of a single donor. (H) Table showing the significance values (P value) from the estimated marginal (EM) means statistics derived from comparisons between DMSO and RUX conditions using a generalized mixed linear model applied to the raw colony counts used to generate Figure 5G. (I) Bar plots showing the fold change in number of colonies produced by HSPCs that were isolated from patients with myelofibrosis and cultured for 7 days in the presence of RUX, normalized to the number of colonies produced by HSPCs cultured for 7 days with DMSO. The data are shown as log₂(fold change) from DMSO. Left showing the fold change in colony numbers in the first round of colony formation (2 weeks in methylcellulose). Right showing the fold change in colony numbers in the second round of colony formation (4 weeks in methylcellulose). Each dot represents the average fold change from each of 4 patients with myelofibrosis, and the bars represent the mean ± SEM. (J) Table showing the significance values (P value) from EM means statistics derived from comparisons between DMSO and RUX conditions using a generalized mixed linear model statistic applied to colony counts used to generate Figure 5I.