Fig. 2. NLK expression contributes to erythroid defects in RPS19-insufficiency.

a Cord blood CD34⁺ progenitors were transduced with lentivirus expressing shRNA against luciferase (shLuc) or RPS19 (shRPS19) co-expressing GFP, along with siRNA targeting NLK (siNLK) co-expressing RFP and a siNLK-resistant NLK (NLK_esc) co-expressing puromycin resistance. GFP⁺RFP⁺ progenitors were differentiated in erythroid media for 15 days prior to counting and assessment for surface expression of (i) CD235 (erythroid), (ii) CD41a (megakaryocyte) and (iii) CD11b (myeloid) cellular markers by flow cytometry. Within each sample, the total number of cells was multiplied by the percentage of cells expressing each differentiation marker and values were normalized and expressed as a percentage of control (shLuc/NT). b Transduced GFP⁺,RFP⁺ cord blood CD34⁺ progenitors were differentiated in methylcellulose for 12–15 days and colonies were scored as either erythroid (i) or myeloid (ii). c Expression of NLK, RPS19, and GAPDH were analyzed by Western blot analysis after 5 days of differentiation. d CD34⁺ progenitors were transduced with shRNA against luciferase or RPS19 and non-targeting or siRNA against NLK. After sorting, samples were split into two groups and either treated with vehicle or SD208 every three days. After 15 days cells were counted and subject to flow cytometry to compare the expansion of maturing CD235⁺ erythroid (i) and CD11b⁺ myeloid cells (ii). 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. 2. Erythroid expansion is depicted in red, megakaryocyte in blue and myeloid in purple. Source data are provided as a Source Data file.