Fig 2: Role of Aβ in the activation of Akt and mammalian target of rapamycin complex 1 (mTORC1). (A) SK-N-MC were incubated with Aβ (5 μM) and NAC (1 mM) for 24 h. Phosphorylated Akt (Thr 308 or Ser 473), Akt and β-actin were detected by western blot. n = 6. (B) Cells were incubated with Aβ for 0–48 h. Phosphorylated mTOR (Ser 2448 and Ser 2481), mTOR and β-actin were detected by western blot. n = 3. (C) Cells were treated with Aβ for 24 h. Protein samples were immunoprecipitated by using mTOR antibody-conjugated protein A/G agarose beads. Immunoprecipitation assay was described in “Materials and Methods” section. Raptor, Rictor and mTOR were detected by western blot. n = 3. (D) Cells were pretreated with LY294002 (20 μM) or Akt inhibitor (20 μM) for 30 min prior to Aβ treatment for 24 h. Cells were blotted with p-mTOR (Ser 2448), mTOR, p-Raptor (Ser 792), Raptor and β-actin specific antibodies. n = 4. Each western blot image was presented as representative image. Quantitative blot data are presented as a mean ± SE. n = 3. *p < 0.05 vs. control, #p < 0.05 vs. Aβ treatment. (E,F) SK-N-MC cells and mouse hippocampal neurons were immunostatined with p-mTOR (Ser 2448) specific antibodies, and visualized by confocal microscopy. Scale bars, 100 μm (magnification, ×400). Data are presented as a mean ± SE. n = 3.

Fig 3: NLK is activated in early erythroid progenitors and requires P53 stabilization in RPS19-insufficiency.a Cord blood CD34+ progenitors were transduced with lentivirus expressing shRNA against luciferase (shLuc) or RPS19 (shRPS19) co-expressing GFP. Cells were also transduced with or without shRNA against p53 co-expressing mCherry. After 36 h, GFP+ and GFP+,mCherry+ cells were differentiated in erythroid media for 8 days, followed by NLK kinase assay (i—NLK: orange, ii—Myb: blue, iii—Raptor: green) and qRT-PCR analysis of RPS19 (iv) and p53 (v) expression. b CD34+ cord blood HSPCs were transduced with control shRNA (shLuc) along with YFP- and CFP-tagged NLK and allowed to differentiate for 3 days, in the presence (i) or absence (ii) of Nutlin-3. Cells were stained for p53 and both p53 and FRET was measured by flow cytometry. For comparison, HSPCs were transduced with shRNA against RPS19 (iii). c Bone marrow mononuclear cells from healthy donors were transduced with YFP- and CFP-NLK and incubated alone (i), or with Nutlin-3 (ii) for 24 h, prior to p53 and FRET analysis. Bone marrow mononuclear cells from three DBA patients were pooled and analyzed simultaneously (iii). d Documentation of the percentage of cells with dimerizing NLK in p53hi and p53low is tabulated and can be compared with NLK in vitro kinase activity. e—green Samples were gated to include non-erythroid CD71-CD235- populations, MEP- and BFU-E-enriched CD71lowCD235- populations, CFU-E-enriched CD71hiCD235- populations and proerythroblast and intermediate erythroblast CD71hiCD235+ populations. Sorted populations were lysed and immunoprecipitated NLK was subjected to in vitro kinase assay examining Raptor phosphorylation as a substrate. Activity was normalized to the non-erythroid control samples. f—green Sorted populations were lysed and immunoprecipitated NLK was subjected to in vitro kinase assay examining Raptor phosphorylation as a substrate. Activity was normalized to the Lin-Kit+Sca+ RPL11+/+ samples. 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. 7. Source data are provided as a Source Data file.

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.

Fig 5: NLK expression is higher in erythroid progenitors and is activated in RPS19-insufficiency.a Transduced CD34+ CB HSPCs were differentiated for 10 days and CD71+ and CD71– fractions were probed by Western blot analysis for NLK using three different NLK antibodies. Equivalent protein was loaded between samples. b—blue Transduced progenitors were differentiated for 15 days and sorted for surface expression of CD235 + , CD41 + and CD11b + population NLK mRNA was assessed by qRT-PCR. c Control and shRPS19-transduced CB progenitors were differentiated for 10 days prior to separation into CD71+ and CD71- populations and assessed for pThr298-NLK phosphorylation by Western blot analysis. d—purple CB CD34 + progenitors were transduced with shRNA against luciferase (shLuc) or RPS19 (shRPS19) along with CFP-NLK and YFP-NLK and differentiated for 10 days. NLK dimerization was quantified by FRET. e—left panel NLK was immunoprecipitated from transduced differentiating CD71+ and CD71- populations after 10 days, and incubated in the presence of ATP, Mg2+ and dephosphorylated NLK (i orange), c-Myb (ii—blue) and Raptor (iii—green) for 30 min at 37 °C. Phosphorylation was detected by a combination of anti-phosphoserine-HRP and anti-phosphothreonine-HRP antibodies, or mouse anti-phosphoserine and anti-mouse-HRP antibodies. e—right panel For comparison, NLK in vitro kinase activity was assessed from CD235+, CD41+ and CD11b+ populations enriched after identical treatments. f A panel of eight small molecule p38 inhibitors were titrated into in vitro kinase assays in the presence of activated NLK or p38 from stimulated Kp53A1 cells. The IC50 for NLK and p38 for each compound was calculated. The data are represented diagrammatically with IC50 values represented as vertical lines along a concentration gradient for NLK and p38. Kinase activity is represented as blue and the extent of inhibition is depicted in white. Our observed values (shown in orange) can be easily compared with documented IC50 values (shown in black) for each compound against each kinase. 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 Figs. 3 and 4. Source data are provided as a Source Data file.