Fig. 8: NLK inhibition increases expansion of erythroid progenitors from human and murine models of DBA.

a Human cord blood CD34⁺ progenitors were transduced with lentivirus co-expressing GFP with shRNA against luciferase (shLuc), RPS19 (shRPS19), or RPL11 (shRPL11). After 36 h GFP⁺ cells were differentiated in erythroid media in the presence or absence of 5 µM SD208 for 15 days. Cells were counted and assessed for cell surface expression of CD235. b Lin-Kit⁺ hematopoietic progenitors were obtained from three mouse embryos expressing tetracycline-inducible shRNA against RPS19 at day E14.5 or three untreated mice, and thee mature RPL11^+/+ or three mature RPL11^+/lox mice treated with tamoxifen for eight weeks. Cells were grown in the presence or absence of doxycycline and/or SD208 for 8 days prior to counting and assessing for Ter119 surface expression. Values were expressed as a percentage relative to untreated controls. c, d Intracellular phosphorylation of NLK at Thr298 was determined by capillary electrophoresis using the Peggy Sue™ Automated Western blotting platform. After lysis, 4 µg of protein from the CD235⁺ population was probed against pThr298-NLK and normalized to GAPDH. Detected NLK phosphorylation is plotted relative to untreated control. e CD34⁺ HSPCs were isolated from three healthy control and three DBA patient mononuclear bone marrow aspirates by magnetic bead sorting and differentiated in the presence or absence of SD208 for 14 days. After counting the total cell population, the ratio of CD235⁺ erythroblasts was determined by flow cytometry and number of CD235⁺ erythroblasts calculated. This was expressed as a percentage of the average number of erythroid cells present in untreated healthy controls. f NLK phosphorylation was assessed by capillary electrophoresis as above. Bars represent means ± SD with individual data points overlaid. Purple depicts untreated controls, yellow depicts controls treated with SD208, red depicts untreated DBA progenitors, while blue depicts DBA progenitors treated with SD208. n = 3 independent experiments performed in triplicate. Statistics: two-tailed Student’s t test, significant *p < 0.05. Also see Supplementary Fig. 8. Source data are provided as a Source Data file.