Unphosphorylated STAT5B enhances HSPC clonogenicity in vitro and HSC maintenance in vivo. (A) Schematic diagram showing the experimental outline of the serial colony replating assays of STAT5-deficient or WT ESLAM (CD45⁺CD150⁺CD48⁻EPCR⁺) HSCs that were transduced with lentivirus containing STAT5B-YF or EV in SCF/IL-11 maintenance cultures. After 3 days of transduction, GFP⁺ living cells were sorted for serial colony replating assays. (B) Bar plots showing the transformed colony numbers derived from WT HSPCs transduced with YF or EV lentivirus. Transformed colony counts = ((colony number × dilution factor) $÷$ starting number of HSCs) (mean ± SEM). The results were from 4 independent experiments and 7 biological replicates. Asterisks indicate the significant differences as determined by Mann-Whitney U tests (∗∗P < .01). (C) Bar plots showing the transformed colony numbers of STAT5-deficient HSPCs transduced with YF or EV lentivirus. Transformed colony counts = ((colony number × dilution factor) $÷$ starting number of HSCs) (mean ± SEM). The results were from 3 independent experiments and 4 biological replicates. Asterisks indicate significant differences as determined by Mann-Whitney U tests (∗P < .05). (D) Schematic diagram showing the outline of the in vivo functional analysis of WT ESLAM HSCs that were transduced with lentivirus containing STAT5B-YF or EV. FACS-sorted WT ESLAM HSCs (CD45.2⁺) were infected with lentivirus and cultured for 3 days in maintenance cultures and then an equal number of GFP⁺ cells were FACS sorted (112 GFP⁺ cells per recipient) and injected into irradiated recipients (CD45.1⁺) with 3 × 10⁵ competitor BMMNCs (CD45.1⁺/CD45.2⁺). Donor chimerism was monitored every 28 days for more than 6 months. Secondary transplantation was then performed using 5 × 10⁶ BMMNCs from primary recipients. BMMNCs from 1 primary recipient were transplanted into up to 2 recipients. (E) Connected line graph showing donor chimerism in primary recipients (mean ± SEM) (experiment described in panel D). Chimerism was derived as the ratio of donor: (donor + competitor). (F) Connected line graph showing donor chimerism in secondary recipients (mean ± SEM) (experiment described in panel D). Chimerism was derived as the ratio of donor/(donor + competitor). (G) Bar plots showing donor chimerism in total BMMNCs and ESLAM HSCs in the BM of the primary transplant recipients (mean ± SEM). (H) Bar plots showing donor chimerism in total BMMNCs and ESLAM HSCs in the BM of the secondary transplant recipients (mean ± SEM). (I) Bar plots showing the ratio of ESLAM HSC donor chimerism to total BMMNC chimerism in primary recipient BM (mean ± SEM; dotted line indicating 1:1 ratio). (J) Bar plots showing the ratio of ESLAM HSC donor chimerism to total BMMNC chimerism in secondary recipient BM (mean ± SEM; dotted line indicating 1:1 ratio). Chimerism was derived as the ratio of donor/(donor + competitor). Asterisks indicate significant differences as determined by Student t tests (∗∗P < .01; ∗P < .05).