Fig. 4. NLK is activated in erythroid progenitors from human and murine models of DBA and DBA patient bone marrow.

a Cord blood CD34⁺ progenitors were transduced with lentivirus co-expressing shRNA against luciferase (shLuc), RPS19 (shRPS19) or RPL11 (shRPL11) and GFP. After 36 h GFP⁺ cells were differentiated in erythroid media for the indicated days prior to immunopurifying NLK for kinase assay measuring in vitro phosphorylation of NLK (i—orange), c-Myb (ii—blue) and Raptor (iii—green) and assessment of RPS19/RPL11 (iv—gray), and NLK (v—blue) expression by qRT-PCR. Solid circles indicate shLuc while open circles indicate shRPS19 or shRPL11. b Lin^-Kit⁺ hematopoietic progenitors were obtained from mouse embryos expressing tetracycline-inducible shRNA against RPS19, at day E14.5. Cells were grown in the presence or absence of doxycycline for 8 days and subjected to NLK kinase assay qRT-PCR for expression of murine RPS19 and NLK (b-left). Lin^-Kit⁺ progenitors were purified from bone marrow of 3 RPL11^+/+ and 3 RPL11^+/lox tamoxifen-treated mice and analyzed for NLK activity by kinase assay, as well as NLK and RPL11 expression by qRT-PCR (b-right). c NLK was immunopurified from 5000 bone marrow mononuclear cells derived from bone marrow aspirates of healthy control and three DBA patients carrying RPS19 mutations. Bars represent means ± SD with individual data points overlaid. n = 3 independent experiments performed in triplicate. Statistics: two-tailed Student’s t test, significant *p < 0.05.Also see Supplementary Fig. 5. Source data are provided as a Source Data file.