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Haematopoietic stem cells require a highly regulated protein synthesis rate

Nature volume 509, pages 49–54 (2014)Cite this article

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Abstract

Many aspects of cellular physiology remain unstudied in somatic stem cells, for example, there are almost no data on protein synthesis in any somatic stem cell. Here we set out to compare protein synthesis in haematopoietic stem cells (HSCs) and restricted haematopoietic progenitors. We found that the amount of protein synthesized per hour in HSCs in vivo was lower than in most other haematopoietic cells, even if we controlled for differences in cell cycle status or forced HSCs to undergo self-renewing divisions. Reduced ribosome function in Rpl24Bst/+ mice further reduced protein synthesis in HSCs and impaired HSC function. Pten deletion increased protein synthesis in HSCs but also reduced HSC function. Rpl24Bst/+ cell-autonomously rescued the effects of Pten deletion in HSCs; blocking the increase in protein synthesis, restoring HSC function, and delaying leukaemogenesis. Pten deficiency thus depletes HSCs and promotes leukaemia partly by increasing protein synthesis. Either increased or decreased protein synthesis impairs HSC function.

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Figure 1: Quantification of protein synthesis in haematopoietic cells in vivo.
Figure 2: Lower rate of OP-Puro incorporation by HSCs does not reflect efflux or proteasomal degradation.
Figure 3: HSCs synthesize less protein than most haematopoietic progenitors, even when undergoing self-renewing divisions.
Figure 4: Rpl24Bst/+ HSCs synthesize less protein and have less capacity to reconstitute irradiated mice.
Figure 5: Rpl24Bst/+ blocks the increase in protein synthesis and restores HSC function after Pten deletion.

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References

  1. Narla, A. & Ebert, B. L. Ribosomopathies: human disorders of ribosome dysfunction. Blood 115, 3196–3205 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Sakamoto, K. M., Shimamura, A. & Davies, S. M. Congenital disorders of ribosome biogenesis and bone marrow failure. Biol. Blood Marrow Transplant. 16, S12–S17 (2010)

    Article  CAS  PubMed  Google Scholar 

  3. Hsieh, A. C. et al. Genetic dissection of the oncogenic mTOR pathway reveals druggable addiction to translational control via 4EBP-eIF4E. Cancer Cell 17, 249–261 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Barna, M. et al. Suppression of Myc oncogenic activity by ribosomal protein haploinsufficiency. Nature 456, 971–975 (2008)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  5. Hsieh, A. C. et al. The translational landscape of mTOR signalling steers cancer initiation and metastasis. Nature 485, 55–61 (2012)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  6. Ruggero, D. & Pandolfi, P. P. Does the ribosome translate cancer? Nature Rev. Cancer 3, 179–192 (2003)

    Article  CAS  Google Scholar 

  7. Jaako, P. et al. Mice with ribosomal protein S19 deficiency develop bone marrow failure and symptoms like patients with Diamond–Blackfan anemia. Blood 118, 6087–6096 (2011)

    Article  CAS  PubMed  Google Scholar 

  8. Wong, C. C., Traynor, D., Basse, N., Kay, R. R. & Warren, A. J. Defective ribosome assembly in Shwachman–Diamond syndrome. Blood 118, 4305–4312 (2011)

    Article  CAS  PubMed  Google Scholar 

  9. Danilova, N., Sakamoto, K. M. & Lin, S. Ribosomal protein S19 deficiency in zebrafish leads to developmental abnormalities and defective erythropoiesis through activation of p53 protein family. Blood 112, 5228–5237 (2008)

    Article  CAS  PubMed  Google Scholar 

  10. Sen, S. et al. The ribosome-related protein, SBDS, is critical for normal erythropoiesis. Blood 118, 6407–6417 (2011)

    Article  CAS  PubMed  Google Scholar 

  11. Payne, E. M. et al. L–Leucine improves the anemia and developmental defects associated with Diamond-Blackfan anemia and del(5q) MDS by activating the mTOR pathway. Blood 120, 2214–2224 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Beatty, K. E. et al. Fluorescence visualization of newly synthesized proteins in mammalian cells. Angew. Chem. Int. Edn Engl. 45, 7364–7367 (2006)

    Article  CAS  Google Scholar 

  13. Nathans, D. Puromycin inhibition of protein synthesis: incorporation of puromycin into peptide chains. Proc. Natl Acad. Sci. USA 51, 585–592 (1964)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  14. Schmidt, E. K., Clavarino, G., Ceppi, M. & Pierre, P. SUnSET, a nonradioactive method to monitor protein synthesis. Nature Methods 6, 275–277 (2009)

    Article  CAS  PubMed  Google Scholar 

  15. Starck, S. R., Green, H. M., Alberola-Ila, J. & Roberts, R. W. A general approach to detect protein expression in vivo using fluorescent puromycin conjugates. Chem. Biol. 11, 999–1008 (2004)

    Article  CAS  PubMed  Google Scholar 

  16. Joseph, N. M. & Morrison, S. J. Toward and understanding of the physiological function of mammalian stem cells. Dev. Cell 173–183 (2005)

  17. Liu, J., Xu, Y., Stoleru, D. & Salic, A. Imaging protein synthesis in cells and tissues with an alkyne analog of puromycin. Proc. Natl Acad. Sci. USA 109, 413–418 (2012)

    Article  ADS  PubMed  Google Scholar 

  18. Kiel, M. J. et al. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 121, 1109–1121 (2005)

    Article  CAS  PubMed  Google Scholar 

  19. Oguro, H., Ding, L. & Morrison, S. J. SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors. Cell Stem Cell 13, 102–116 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Zhou, S. et al. Bcrp1 gene expression is required for normal numbers of side population stem cells in mice, and confers relative protection to mitoxantrone in hematopoietic cells in vivo . Proc. Natl Acad. Sci. USA 99, 12339–12344 (2002)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  21. Ciechanover, A., Finley, D. & Varshavsky, A. Ubiquitin dependence of selective protein degradation demonstrated in the mammalian cell cycle mutant ts85. Cell 37, 57–66 (1984)

    Article  CAS  PubMed  Google Scholar 

  22. Luker, G. D., Pica, C. M., Song, J., Luker, K. E. & Piwnica-Worms, D. Imaging 26S proteasome activity and inhibition in living mice. Nature Med. 9, 969–973 (2003)

    Article  CAS  PubMed  Google Scholar 

  23. Gingras, A. C., Raught, B. & Sonenberg, N. Regulation of translation initiation by FRAP/mTOR. Genes Dev. 15, 807–826 (2001)

    Article  CAS  PubMed  Google Scholar 

  24. Wek, R. C., Jiang, H. Y. & Anthony, T. G. Coping with stress: eIF2 kinases and translational control. Biochem. Soc. Trans. 34, 7–11 (2006)

    Article  CAS  PubMed  Google Scholar 

  25. Morrison, S. J., Wright, D. & Weissman, I. L. Cyclophosphamide/granulocyte colony-stimulating factor induces hematopoietic stem cells to proliferate prior to mobilization. Proc. Natl Acad. Sci. USA 94, 1908–1913 (1997)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  26. Oliver, E. R., Saunders, T. L., Tarle, S. A. & Glaser, T. Ribosomal protein L24 defect in belly spot and tail (Bst), a mouse Minute . Development 131, 3907–3920 (2004)

    Article  CAS  PubMed  Google Scholar 

  27. Fumagalli, S. & Thomas, G. The role of p53 in ribosomopathies. Semin. Hematol. 48, 97–105 (2011)

    Article  CAS  PubMed  Google Scholar 

  28. Barkić, M. et al. The p53 tumor suppressor causes congenital malformations in Rpl24-deficient mice and promotes their survival. Mol. Cell. Biol. 29, 2489–2504 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Lee, J. Y. et al. mTOR activation induces tumor suppressors that inhibit leukemogenesis and deplete hematopoietic stem cells after Pten deletion. Cell Stem Cell 7, 593–605 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Magee, J. A. et al. Temporal changes in PTEN and mTORC2 regulation of hematopoietic stem cell self-renewal and leukemia suppression. Cell Stem Cell 11, 415–428 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Yilmaz, O. H. et al. Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells. Nature 441, 475–482 (2006)

    Article  ADS  CAS  PubMed  Google Scholar 

  32. Kalaitzidis, D. et al. mTOR Complex 1 plays critical roles in hematopoiesis and pten-loss-evoked leukemogenesis. Cell Stem Cell 11, 429–439 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Zhang, J. et al. PTEN maintains haematopoietic stem cells and acts in lineage choice and leukaemia prevention. Nature 441, 518–522 (2006)

    Article  ADS  CAS  PubMed  Google Scholar 

  34. Zinzalla, V., Stracka, D., Oppliger, W. & Hall, M. N. Activation of mTORC2 by association with the ribosome. Cell 144, 757–768 (2011)

    Article  CAS  PubMed  Google Scholar 

  35. Guo, W. et al. Multi-genetic events collaboratively contribute to Pten-null leukaemia stem-cell formation. Nature 453, 529–533 (2008)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  36. Ruggero, D. Translational control in cancer etiology. Cold Spring Harb. Perspect. Biol. 5, a012336 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Dahlberg, A., Delaney, C. & Bernstein, I. D. Ex vivo expansion of human hematopoietic stem and progenitor cells. Blood 117, 6083–6090 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Huang, J., Nguyen-McCarty, M., Hexner, E. O., Danet-Desnoyers, G. & Klein, P. S. Maintenance of hematopoietic stem cells through regulation of Wnt and mTOR pathways. Nature Med. 18, 1778–1785 (2012)

    Article  CAS  PubMed  Google Scholar 

  39. Vilchez, D. et al. Increased proteasome activity in human embryonic stem cells is regulated by PSMD11. Nature 489, 304–308 (2012)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  40. Vilchez, D. et al. RPN-6 determines C. elegans longevity under proteotoxic stress conditions. Nature 489, 263–268 (2012)

    Article  ADS  CAS  PubMed  Google Scholar 

  41. Signer, R. A. & Morrison, S. J. Mechanisms that regulate stem cell aging and life span. Cell Stem Cell 12, 152–165 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Groszer, M. et al. PTEN negatively regulates neural stem cell self-renewal by modulating G0-G1 cell cycle entry. Proc. Natl Acad. Sci. USA 103, 111–116 (2006)

    Article  ADS  CAS  PubMed  Google Scholar 

  43. Kühn, R., Schwenk, F., Aguet, M. & Rajewsky, K. Inducible gene targeting in mice. Science 269, 1427–1429 (1995)

    Article  ADS  PubMed  Google Scholar 

  44. Jonker, J. W. et al. The breast cancer resistance protein protects against a major chlorophyll-derived dietary phototoxin and protoporphyria. Proc. Natl Acad. Sci. USA 99, 15649–15654 (2002)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  45. Jacks, T. et al. Tumor spectrum analysis in p53-mutant mice. Curr. Biol. 4, 1–7 (1994)

    Article  CAS  PubMed  Google Scholar 

  46. Akashi, K., Traver, D., Miyamoto, T. & Weissman, I. L. A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature 404, 193–197 (2000)

    Article  ADS  CAS  PubMed  Google Scholar 

  47. Hardy, R. R., Carmack, C. E., Shinton, S. A., Kemp, J. D. & Hayakawa, K. Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow. J. Exp. Med. 173, 1213–1225 (1991)

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

S.J.M. is a Howard Hughes Medical Institute Investigator, the Mary McDermott Cook Chair in Pediatric Genetics, and the director of the Hamon Laboratory for Stem Cells and Cancer. This work was supported by the Cancer Prevention and Research Institute of Texas and the National Institute on Aging (R37 AG024945). R.A.J.S. was supported by fellowships from the Leukemia & Lymphoma Society (5541-11) and the Canadian Institutes of Health Research (MFE-106993). J.A.M. was supported by the UT Southwestern K12 Pediatrics Training Grant (K12-HD068369). We thank A. Pineda, K. Cowan, E. Daniel, M. Acar, H. Oguro, J. Peyer, K. Rajagopalan, M. Agathocleous and E. Piskounova for technical support and advice; N. Loof and the Moody Foundation Flow Cytometry Facility, L. Hynan and J. Reisch for advice regarding statistics; J. Shelton for histology; and R. Coolon, S. Manning, M. Gross and K. Correll for mouse colony management.

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  1. Department of Pediatrics, Howard Hughes Medical Institute, Children’s Research Institute, University of Texas Southwestern Medical Center, Dallas, 75390, Texas, USA

    Robert A. J. Signer, Jeffrey A. Magee & Sean J. Morrison

  2. Department of Cell Biology, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Adrian Salic

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Contributions

R.A.J.S. conceived the project. A.S. developed the OP-Puro reagent and provided advice in the early stages of the project. R.A.J.S. performed all of the experiments with the exception of the western blot analyses in Figs 2e and 5a, d, and Extended Data Fig. 5j, which were performed by J.A.M. R.A.J.S., J.A.M. and S.J.M. designed the experiments and interpreted results. R.A.J.S. and S.J.M. wrote the manuscript.

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Correspondence to Sean J. Morrison.

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Extended data figures and tables

Extended Data Figure 1 Isolation of haematopoietic progenitor cell populations by flow cytometry and histograms showing protein synthesis in vivo relative to HSCs from the same mice.

a–e, One hour after OP-Puro administration to mice we observed no effect on bone marrow cellularity (one femur and one tibia (a); n = 7 PBS treated and n = 9 OP-Puro treated mice) or the frequencies of CD150+CD48-LSK HSCs (b; n = 4 PBS treated, n = 6 OP-Puro treated mice), annexin V+ bone marrow cells (c; n = 4 PBS treated mice, n = 6 OP-Puro treated mice), annexin V+ HSCs (d; n = 4 PBS treated mice, n = 6 OP-Puro treated mice), or HSCs in S/G2/M phase of the cell cycle (e; n = 3 mice per treatment; a–e each reflect two or three independent experiments). f–i, Representative flow-cytometry plots showing the markers and gating strategies used to isolate CMPs46, GMPs46, and MEPs46 (f), pro-B47, pre-B47 and IgM+ B cells (g), Gr-1+ myeloid cells (h), CD3+ T cells (h) and CD71+Ter119+ erythroid progenitors (i). Each panel also shows OP-Puro incorporation histograms for each cell population relative to HSCs after 1 h of OP-Puro incorporation in vivo. The level of background fluorescence from PBS treated controls is overlaid in black. j, Data from Fig. 1h showing protein synthesis in various haematopoietic-cell populations relative to unfractionated bone marrow cells on a log2 scale (n = 15 mice from 9 independent experiments). All data represent mean ± s.d. Two-tailed Student’s t-tests were used to assess statistical significance in a–e. The statistical significance of differences relative to HSCs in j was assessed using a repeated-measures one-way ANOVA followed by Dunnett’s test for multiple comparisons. Asterisks indicate statistical comparison to HSCs (*P < 0.05, **P < 0.01, ***P < 0.001).

Extended Data Figure 2 OP-Puro-containing polypeptides are not degraded within 30 min, the degradation that occurs over 24 h is blocked by bortezomib, and OP-Puro administration does not induce cell death.

a, OP-Puro fluorescence in haematopoietic cells after 1 h of OP-Puro administration in vivo followed by a 30-min ex vivo incubation on ice or at 37 °C (n = 11 mice from 4 independent experiments). b, OP-Puro fluorescence in haematopoietic cells 24 h after OP-Puro administration in vivo. Treatment with bortezomib 1 h before OP-Puro administration increased OP-Puro fluorescence in every cell population 24 h later (n = 3 independent experiments; total number of mice per treatment are shown in the panel). c, Frequency of annexin V+ cells in each cell population 1 h after OP-Puro administration in vivo relative to the same cells from untreated mice (n = 7 mice per treatment from 2 independent experiments). All data represent mean ± s.d. To assess the statistical significance of treatment effects within the same cell population we performed two-tailed Student’s t-tests (*P < 0.05, **P < 0.01, ***P < 0.001). To assess the statistical significance of differences between HSCs and other cell populations in a, we performed a repeated-measures one-way ANOVA followed by Dunnett’s test for multiple comparisons (†P < 0.05, ††P < 0.01, †††P < 0.001).

Extended Data Figure 3 Cyclophosphamide and GCSF treatment drives certain cells into cycle and increases protein synthesis.

a, Frequency of dividing cells in S/G2/M phases of the cell cycle (>2N (>diploid) DNA content; n = 5 mice from 3 independent experiments). b, Total protein isolated from 50,000 unfractionated bone marrow cells or Gr-1+ cells in G0/G1 or S/G2/M measured by BCA assay (n = 3). c, Total RNA content in 15,000 cells from each stem- or progenitor-cell population (n = 3 mice). d, The frequency of cycling (KI-67+) HSCs increased dramatically after treatment with cyclophosphamide (Cy) and GCSF (n = 5 untreated mice and n = 6 mice treated with cyclophosphamide and GCSF, from 2 independent experiments, P < 0.001). e, Frequency of KI-67+ cells in haematopoietic-cell populations before and after treatment with cyclophosphamide and GCSF (n = 5 untreated mice and n = 6 mice treated with cyclophosphamide and GCSF for BM, HSC and MPP, n = 3 mice per treatment for other cell populations). f, g, Protein synthesis in G0/G1 and S/G2/M cells from untreated mice or mice treated with cyclophosphamide followed by two days of GCSF (n = 10 mice per treatment from 6 independent experiments). These data are the same as shown in Fig. 3b, d, e, shown together in this panel for comparison. The data are plotted on a linear scale in f and on a log2 scale in g. All data represent mean ± s.d. To assess the statistical significance of treatment effects within the same cell population (b, e–g) we performed two-tailed Student’s t-tests (*P < 0.05, **P < 0.01, ***P < 0.001). To assess the statistical significance of differences between HSCs and each other cell population (a, c, f, g) we performed a repeated-measures one-way ANOVA followed by Dunnett’s test for multiple comparisons (†P < 0.05, ††P < 0.01, †††P < 0.001).

Extended Data Figure 4 Differences in protein synthesis among haematopoietic stem and progenitor cells are not fully explained by differences in cell division, cell diameter, pS6 levels, rRNA or total RNA content.

a–f, Scatter plots show the relative rates of protein synthesis (per hour) in each cell population (from Fig. 1h) plotted against the frequency of dividing cells (a; from Extended Data Fig. 3a), cell diameter (b; from Fig. 3f), 18S rRNA content (c; from Fig. 3g), 28S rRNA content (d; from Fig. 3g), total RNA content (e; from Extended Data Fig. 3c) and pS6 levels (f; from Fig. 5a normalized to β-actin). For each parameter, regressions were performed using all populations excluding HSCs and 95% confidence intervals were determined. R2 values are shown in each plot. Rates of protein synthesis are plotted on a linear scale (left panels) and on a log2 scale (right panels). Note that HSCs were outliers with respect to each regression. CD150+CD48−LSK cells were used to determine HSC rates of protein synthesis, cell diameter and percentage S/G2/M, and CD48−LSK cells (HSCs and MPPs) were used to determine 18S, 28S, total RNA and pS6 levels (as these measurements required more cells). All data represent mean ± s.d.

Extended Data Figure 5 Rpl24Bst/+ mice have normal frequencies of lymphoid and myeloid lineage progenitors and do not express increased p53 or p21Cip1 in adult haematopoietic cells.

a, Bone marrow (2 femurs and 2 tibias; n = 5 wild-type and n = 6 Rpl24Bst/+ mice from 4 experiments), spleen (n = 3 wild-type and n = 4 Rpl24Bst/+ mice from 2 experiments), and thymus cellularity (n = 3 wild-type and n = 4 Rpl24Bst/+ mice from 2 experiments). b, White blood cell, red blood cell and platelet counts (n = 5 wild-type and n = 6 Rpl24Bst/+ mice from 4 experiments). c–f, The frequencies of B (c), myeloid (d) and T (e) lineage cells in the bone marrow and spleen (f) of Rpl24Bst/+ and control mice (n = 3 wild-type and n = 4 Rpl24Bst/+ mice from 2 experiments). g, h, The frequencies of T-lineage progenitors in the thymus of Rpl24Bst/+ and control mice (n = 3 wild-type and n = 4 Rpl24Bst/+ mice from 2 experiments). Double negative (DN)1 and early T-lineage progenitor (ETP) cells were CD4−CD8−CD44+CD25−; DN2 cells were CD4−CD8−CD44+CD25+; DN3 cells were CD4−CD8−CD44−CD25+; and DN4 cells were CD4−CD8−CD44-CD25−. i, The frequencies of annexin V+ HSCs and MPPs in Rpl24Bst/+ versus littermate control mice (n = 3 wild-type and n = 4 Rpl24Bst/+ mice from 3 experiments). j, Western blot analysis for Rpl24 and β-actin using 30,000 cells from each haematopoietic-cell population. Differences in β-actin between lanes represent differences in β-actin content per cell (one representative blot from two independent experiments). k, Donor- B-cell, T-cell and myeloid-cell engraftment when 5 × 105 donor bone marrow cells were transplanted along with 5 × 105 recipient bone marrow cells into irradiated recipient mice (n = 4 independent experiments with a total of 17 recipients for wild-type cells and 20 for Rpl24Bst/+ cells). These transplant recipients are the same as those shown in Fig. 4e. l, The frequency of donor cells in the bone marrow 20 h after transplanting 1 × 105 donor LSK cells from Rpl24Bst/+ or wild-type control mice into irradiated recipient mice (n = 3 recipients per donor). The horizontal line represents the level of background detected in an untransplanted control. m, Western blot analysis for p53 using 5 × 105 Lineage− bone marrow cells from wild-type or Rpl24Bst/+ mice, or 5 × 105 bone marrow cells from a p53−/− mouse (one representative blot from two experiments). n, Western blot analysis for p21Cip1 using 285,000 LSK cells from the bone marrow of adult wild-type or Rpl24Bst/+ mice, or 142,500 LSK cells from a wild-type mouse that received 540 rad of total body irradiation 3 to 4 h before being euthanized (one representative blot from three independent experiments). All data represent mean ± s.d. Two-tailed Student’s t-tests were used to assess statistical significance (*P < 0.05, **P < 0.01, ***P < 0.001).

Extended Data Figure 6 Rpl24Bst/+ and Pten-deficient progenitors form colonies with normal cellularity but Rpl24Bst/+ impairs the development of haematopoietic neoplasms after Pten deletion.

a, b, The percentage of HSCs (a) or bone marrow cells (b) that formed colonies in methylcellulose within 14 days of culture (n = 3 mice per genotype in 3 independent experiments with 16 HSCs or 3,200 bone marrow cells tested per mouse per experiment). c, d, The average number of cells per granulocyte-monocyte (GM) or granulocyte, erythrocyte, monocyte, megakaryocyte (GEMM) colony derived from single HSCs (c) or bone marrow cells plated at clonal density (d) (n = 4 independent experiments). e, The average number of cells per GM or GEMM colony derived from individual HSCs of the indicated genotypes 15 days after plating (n = 2 independent experiments). f, Frequency of annexin V+ CD4+CD8+ thymocytes (n = 5 independent experiments). g, Representative histograms of OP-Puro fluorescence in HSCs of the indicated genotypes. h, Mass of spleens and thymuses 2 weeks after pIpC administration (n = 7 independent experiments). i, Representative photographs of thymuses 2 weeks after pIpC administration to wild-type, Mx1-Cre; Ptenfl/fl, Rpl24Bst/+ and Mx1-Cre; Ptenfl/fl; Rpl24Bst/+ mice. j, HSCs in the spleen 2 weeks after pIpC administration (n = 7 independent experiments). k, Haematoxylin and eosin stained spleen and thymus sections from mice 2 weeks after pIpC administration or when they were killed owing to illness. All data represent mean ± s.d. In a–d and f, two-tailed Student’s t-tests were used to assess statistical significance relative to wild-type; *P < 0.05, **P < 0.01. To assess statistical significance in e we performed a one-way ANOVA followed by Tukey’s t-tests for multiple comparisons (relative to wild-type, *P < 0.05; ***P < 0.001; and relative to p53+/− , †P < 0.05, ††P < 0.01). To compare the statistical significance of differences among genotypes in h and j we performed a one-way ANOVA followed by Dunnett’s test for multiple comparisons relative to Pten-deficient (**P < 0.01, ***P < 0.001).

Extended Data Figure 7 Rpl24Bst/+ and Pten-deficient MPPs have relatively normal reconstituting activity.

a, b, 100 donor CD150−CD48−LSK MPPs from Mx1-Cre; Ptenfl/fl versus control mice (a; n = 3 independent experiments with a total of 13 recipients per genotype) or Rpl24Bst/+ versus control mice (b; n = 3 independent experiments with a total of 14 recipients of wild-type cells and 13 recipients of Rpl24Bst/+ cells) were transplanted along with 3 × 105 recipient-type bone marrow cells into irradiated recipient mice. Donor-cell engraftment levels in the peripheral blood were assessed at 3, 5 and 7 weeks after transplantation. All data represent mean ± s.d. Two-tailed Student’s t-tests were used to assess statistical significance relative to wild-type; *P < 0.05.

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Signer, R., Magee, J., Salic, A. et al. Haematopoietic stem cells require a highly regulated protein synthesis rate. Nature 509, 49–54 (2014). https://doi.org/10.1038/nature13035

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