NF-κB activation impairs somatic cell reprogramming in ageing

  • This article was retracted on 17 December 2018

Abstract

Ageing constitutes a critical impediment to somatic cell reprogramming. We have explored the regulatory mechanisms that constitute age-associated barriers, through derivation of induced pluripotent stem cells (iPSCs) from individuals with premature or physiological ageing. We demonstrate that NF-κB activation blocks the generation of iPSCs in ageing. We also show that NF-κB repression occurs during cell reprogramming towards a pluripotent state. Conversely, ageing-associated NF-κB hyperactivation impairs the generation of iPSCs by eliciting the reprogramming repressor DOT1L, which reinforces senescence signals and downregulates pluripotency genes. Genetic and pharmacological NF-κB inhibitory strategies significantly increase the reprogramming efficiency of fibroblasts from Néstor–Guillermo progeria syndrome and Hutchinson–Gilford progeria syndrome patients, as well as from normal aged donors. Finally, we demonstrate that DOT1L inhibition in vivo extends lifespan and ameliorates the accelerated ageing phenotype of progeroid mice, supporting the interest of studying age-associated molecular impairments to identify targets of rejuvenation strategies.

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Figure 1: BAF deficiency impairs cellular reprogramming into iPSCs.
Figure 2: Differentiation defects in NGPS MSCs.
Figure 3: NF-κB hyperactivation in NGPS fibroblasts constitutes a barrier to cell reprogramming.
Figure 4: Mechanisms of NF-κB reprogramming barrier.
Figure 5: DOT1L is a prominent effector of NF-κB signalling.
Figure 6: NF-κB-driven reprogramming impediment in HGPS.
Figure 7: Effect of NF-κB blockade in cell reprogramming of normal ageing.
Figure 8: DOT1L inhibition extends longevity and prevents age-associated alterations.

Change history

  • 04 October 2018

    Editor's Note: We would like to alert readers that the reliability of data presented in this manuscript has been the subject of criticisms, which we are currently considering. We will publish an update once our investigation is complete.

References

  1. 1

    Mahmoudi, S. & Brunet, A. Aging and reprogramming: a two-way street. Curr. Opin. Cell Biol. 24, 744–756 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  2. 2

    Lopez-Otin, C., Blasco, M. A., Partridge, L., Serrano, M. & Kroemer, G. The hallmarks of aging. Cell 153, 1194–1217 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  3. 3

    Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

    CAS  Article  Google Scholar 

  4. 4

    Robinton, D. A. & Daley, G. Q. The promise of induced pluripotent stem cells in research and therapy. Nature 481, 295–305 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  5. 5

    Li, H. et al. The Ink4/Arf locus is a barrier for iPS cell reprogramming. Nature 460, 1136–1139 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. 6

    Wang, B. et al. Reprogramming efficiency and quality of induced Pluripotent Stem Cells (iPSCs) generated from muscle-derived fibroblasts of mdx mice at different ages. PLoS Curr. 3, RRN1274 (2011).

    PubMed  PubMed Central  Article  Google Scholar 

  7. 7

    Kim, K. et al. Epigenetic memory in induced pluripotent stem cells. Nature 467, 285–290 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  8. 8

    Marion, R. M. et al. A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity. Nature 460, 1149–1153 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. 9

    Banito, A. et al. Senescence impairs successful reprogramming to pluripotent stem cells. Genes Dev. 23, 2134–2139 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. 10

    Marion, R. M. et al. Telomeres acquire embryonic stem cell characteristics in induced pluripotent stem cells. Cell Stem Cell 4, 141–154 (2009).

    CAS  PubMed  Article  Google Scholar 

  11. 11

    Suhr, S. T. et al. Mitochondrial rejuvenation after induced pluripotency. PLoS ONE 5, e14095 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. 12

    Kawamura, T. et al. Linking the p53 tumour suppressor pathway to somatic cell reprogramming. Nature 460, 1140–1144 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. 13

    Lapasset, L. et al. Rejuvenating senescent and centenarian human cells by reprogramming through the pluripotent state. Genes Dev. 25, 2248–2253 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. 14

    Rando, T. A. & Chang, H. Y. Aging, rejuvenation, and epigenetic reprogramming: resetting the aging clock. Cell 148, 46–57 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  15. 15

    Freije, J. M. & Lopez-Otin, C. Reprogramming aging and progeria. Curr. Opin. Cell Biol. 24, 757–764 (2012).

    CAS  PubMed  Article  Google Scholar 

  16. 16

    Schreiber, K. H. & Kennedy, B. K. When lamins go bad: nuclear structure and disease. Cell 152, 1365–1375 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. 17

    Osorio, F. G. et al. Splicing-directed therapy in a new mouse model of human accelerated aging. Sci. Transl. Med. 3, 106ra107 (2011).

    PubMed  Article  CAS  Google Scholar 

  18. 18

    Gordon, L. B., Rothman, F. G., Lopez-Otin, C. & Misteli, T. Progeria: a paradigm for translational medicine. Cell 156, 400–407 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. 19

    Miller, J. D. et al. Human iPSC-based modeling of late-onset disease via progerin-induced aging. Cell Stem Cell 13, 691–705 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. 20

    Zhang, J. et al. A human iPSC model of Hutchinson Gilford Progeria reveals vascular smooth muscle and mesenchymal stem cell defects. Cell Stem Cell 8, 31–45 (2011).

    CAS  PubMed  Article  Google Scholar 

  21. 21

    Liu, G. H. et al. Recapitulation of premature ageing with iPSCs from Hutchinson-Gilford progeria syndrome. Nature 472, 221–225 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. 22

    Nissan, X. et al. Unique preservation of neural cells in Hutchinson- Gilford progeria syndrome is due to the expression of the neural-specific miR-9 microRNA. Cell Rep. 2, 1–9 (2012).

    CAS  PubMed  Article  Google Scholar 

  23. 23

    Scaffidi, P. & Misteli, T. Lamin A-dependent nuclear defects in human aging. Science 312, 1059–1063 (2006).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  24. 24

    Puente, X. S. et al. Exome sequencing and functional analysis identifies BANF1 mutation as the cause of a hereditary progeroid syndrome. Am. J. Hum. Genet. 88, 650–656 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  25. 25

    Cabanillas, R. et al. Nestor-Guillermo progeria syndrome: a novel premature aging condition with early onset and chronic development caused by BANF1 mutations. Am. J. Med. Genet. A 155A, 2617–2625 (2011).

    PubMed  Article  CAS  Google Scholar 

  26. 26

    Margalit, A., Brachner, A., Gotzmann, J., Foisner, R. & Gruenbaum, Y. Barrier-to-autointegration factor—a BAFfling little protein. Trends Cell Biol. 17, 202–208 (2007).

    CAS  PubMed  Article  Google Scholar 

  27. 27

    Buganim, Y., Faddah, D. A. & Jaenisch, R. Mechanisms and models of somatic cell reprogramming. Nat. Rev. Genet. 14, 427–439 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. 28

    Liang, G. & Zhang, Y. Genetic and epigenetic variations in iPSCs: potential causes and implications for application. Cell Stem Cell 13, 149–159 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  29. 29

    Soldner, F. & Jaenisch, R. Medicine. iPSC disease modeling. Science 338, 1155–1156 (2012).

    PubMed  Article  Google Scholar 

  30. 30

    Smahi, A. et al. Genomic rearrangement in NEMO impairs NF-κB activation and is a cause of incontinentia pigmenti. The International Incontinentia Pigmenti (IP) Consortium. Nature 405, 466–472 (2000).

    CAS  PubMed  Article  Google Scholar 

  31. 31

    Luningschror, P., Stocker, B., Kaltschmidt, B. & Kaltschmidt, C. miR-290 cluster modulates pluripotency by repressing canonical NF-κB signaling. Stem Cells 30, 655–664 (2012).

    PubMed  Article  CAS  Google Scholar 

  32. 32

    Takase, O. et al. The role of NF-κB signaling in the maintenance of pluripotency of human induced pluripotent stem cells. PLoS ONE 8, e56399 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. 33

    Torres, J. & Watt, F. M. Nanog maintains pluripotency of mouse embryonic stem cells by inhibiting NF-κB and cooperating with Stat3. Nat. Cell Biol. 10, 194–201 (2008).

    CAS  PubMed  Article  Google Scholar 

  34. 34

    Kuo, H. P. et al. Epigenetic roles of MLL oncoproteins are dependent on NF-κB. Cancer Cell 24, 423–437 (2013).

    CAS  PubMed  Article  Google Scholar 

  35. 35

    Wang, H. et al. NF-κB regulation of YY1 inhibits skeletal myogenesis through transcriptional silencing of myofibrillar genes. Mol. Cell. Biol. 27, 4374–4387 (2007).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. 36

    Onder, T. T. et al. Chromatin-modifying enzymes as modulators of reprogramming. Nature 483, 598–602 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. 37

    Coppe, J. P., Desprez, P. Y., Krtolica, A. & Campisi, J. The senescence-associated secretory phenotype: the dark side of tumor suppression. Annu. Rev. Pathol. 5, 99–118 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. 38

    Pendas, A. M. et al. Defective prelamin A processing and muscular and adipocyte alterations in Zmpste24 metalloproteinase-deficient mice. Nat. Genet. 31, 94–99 (2002).

    CAS  PubMed  Article  Google Scholar 

  39. 39

    Varela, I. et al. Combined treatment with statins and aminobisphosphonates extends longevity in a mouse model of human premature aging. Nat. Med. 14, 767–772 (2008).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  40. 40

    Osorio, F. G. et al. Nuclear lamina defects cause ATM-dependent NF-κB activation and link accelerated aging to a systemic inflammatory response. Genes Dev. 26, 2311–2324 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  41. 41

    Cau, P. et al. Nuclear matrix, nuclear envelope and premature aging syndromes in a translational research perspective. Semin. Cell Dev. Biol. 29, 125–147 (2014).

    CAS  PubMed  Article  Google Scholar 

  42. 42

    Adler, A. S. et al. Motif module map reveals enforcement of aging by continual NF-κB activity. Genes Dev. 21, 3244–3257 (2007).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  43. 43

    Tilstra, J. S. et al. NF-κB inhibition delays DNA damage-induced senescence and aging in mice. J. Clin. Invest. 122, 2601–2612 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  44. 44

    Jurk, D. et al. Chronic inflammation induces telomere dysfunction and accelerates ageing in mice. Nat. Commun. 2, 4172 (2014).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  45. 45

    Baker, D. J. et al. Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature 479, 232–236 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  46. 46

    Kawahara, T. L. et al. SIRT6 links histone H3 lysine 9 deacetylation to NF-κB-dependent gene expression and organismal life span. Cell 136, 62–74 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  47. 47

    Osorio, F. G. et al. Nuclear envelope alterations generate an aging-like epigenetic pattern in mice deficient in Zmpste24 metalloprotease. Aging Cell 9, 947–957 (2010).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  48. 48

    Han, S. & Brunet, A. Histone methylation makes its mark on longevity. Trends Cell Biol. 22, 42–49 (2012).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  49. 49

    De Vos, D. et al. Progressive methylation of ageing histones by Dot1 functions as a timer. EMBO Rep. 12, 956–962 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  50. 50

    Chien, Y. et al. Control of the senescence-associated secretory phenotype by NF-κB promotes senescence and enhances chemosensitivity. Genes Dev. 25, 2125–2136 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  51. 51

    Baker, D. J., Weaver, R. L. & van Deursen, J. M. p21 both attenuates and drives senescence and aging in BubR1 progeroid mice. Cell Rep. 3, 1164–1174 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  52. 52

    Krishnan, V. et al. Histone H4 lysine 16 hypoacetylation is associated with defective DNA repair and premature senescence in Zmpste24-deficient mice. Proc. Natl Acad. Sci. USA 108, 12325–12330 (2011).

    CAS  PubMed  Article  Google Scholar 

  53. 53

    Daigle, S. R. et al. Potent inhibition of DOT1L as treatment of MLL-fusion leukemia. Blood 122, 1017–1025 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  54. 54

    Varela, I. et al. Accelerated ageing in mice deficient in Zmpste24 protease is linked to p53 signalling activation. Nature 437, 564–568 (2005).

    CAS  PubMed  Article  Google Scholar 

  55. 55

    Soria-Valles, C. et al. Somatic cell reprogramming using NF-κB inhibitory strategies. Nat. Protoc. Exch. (2015) http://dx.doi.org/10.1038/protex.2015.057

  56. 56

    Sanchez, L. et al. Enrichment of human ESC-derived multipotent mesenchymal stem cells with immunosuppressive and anti-inflammatory properties capable to protect against experimental inflammatory bowel disease. Stem Cells 29, 251–262 (2011).

    CAS  PubMed  Article  Google Scholar 

  57. 57

    Liu, B. et al. Resveratrol rescues SIRT1-dependent adult stem cell decline and alleviates progeroid features in laminopathy-based progeria. Cell Metab. 16, 738–750 (2012).

    CAS  PubMed  Article  Google Scholar 

  58. 58

    Xu, C. et al. Immortalized fibroblast-like cells derived from human embryonic stem cells support undifferentiated cell growth. Stem Cells 22, 972–980 (2004).

    CAS  PubMed  Article  Google Scholar 

  59. 59

    Doi, A. et al. Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts. Nat. Genet. 41, 1350–1353 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

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Acknowledgements

We thank R. M. Schmid, H. Algül, T. Schöler, A. R. Folgueras, X. S. Puente, A. A. Ferrando, M. Serrano, H. Li, A. López-Soto, C. Bárcena, M. Gupta and D. Robinton for helpful comments and advice. We also thank F. Rodríguez and M. Roldán for excellent technical assistance, and the Servicio de Histopatología (IUOPA) for histological studies. This work was supported by grants from Ministerio de Economía y Competitividad-Spain (to C.L.-O., P.M. and C.B.), Gobierno del Principado de Asturias and Instituto de Salud Carlos III (RTICC), Spain. The Instituto Universitario de Oncología is supported by Fundación Bancaria Caja de Ahorros de Asturias. C.B. is a recipient of a ‘Miguel Servet’ fellowship from the ISCIII-FIS. P.M. is a member of the Spanish Cell Therapy Network (Tercel). C.L.-O. is an Investigator of the Botin Foundation supported by Banco Santander through its Santander Universities Global Division. This paper is dedicated to the memory of Néstor M.O.

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C.S.-V. and F.G.O. performed experimental work, data interpretation and preparation of the manuscript. A.G.-F., C.B. and J.I.M.-S. performed experimental work. A.D.L.A., P.M. and G.Q.D. provided critical materials and participated in the preparation of the manuscript. J.M.P.F. and C.L.-O. supervised research and project planning, data interpretation and preparation of the manuscript. All authors discussed the results and implications and commented on the manuscript at all stages.

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Correspondence to José M. P. Freije or Carlos López-Otín.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Reversal of NGPS alterations through expression of wild-type BANF1.

(a) qRT–PCR of OCT4, SOX2, KLF4 and c-MYC in control and NGPS fibroblasts at day 3 of reprogramming. n = 3 independent experiments. (b) Control fibroblasts were transduced with a BANF1-specific shRNA. qRT–PCR of BANF1 mRNA was performed in pLKO.1 and shBANF1 transduced fibroblasts. mRNA mean relative values are represented. n = 3 independent experiments, P = 0.002. (c) BANF1 down-regulation impedes cell reprogramming. pLKO.1 and shBANF1 transduced-fibroblasts were reprogrammed. Plot represents the number of Tra-1-60 positive colonies. n = 3 independent experiments, P = 0.001. (d) Scheme depicting the strategy followed for genome editing of NGPS-iPSCs using CRISPR-Cas9 system. The purpose of this experiment was introducing through homologous-directed repair the BANF1 wild-type sequence in NGPS-iPSCs. (e) Two representative clones from NGPS-iPSCs were analysed by PCR to detect the presence of the recombinant allele. Note that recombinant allele c.34G expression in NGPS-iPSCs notably increased the levels of BAF. Western blot analysis of BAF in edited NGPS fibroblasts (left). Western-blot analysis of BAF in control and NGPS fibroblasts transduced with a retroviral vector containing wild-type BANF1 transgene (right). A representative image is shown of 3 independent experiments. (f) Representative analysis of nuclear envelope abnormalities in fibroblasts derived from NGPS-corrected iPSCs. n = 100 cells assessed in 3 independent preparations, P = 0.001 both comparisons. (g) NGPS fibroblasts transduced with wild-type BANF1 were reprogrammed into iPSCs. The plot represents the number of Tra-1-60 positive colonies. n = 3 independent experiments, P = 0.001. Error bars indicate SEM (P < 0.01, two-tailed Student’s t test). Unprocessed original scans of blots are shown in Supplementary Fig. 9.

Supplementary Figure 2 Detailed analysis of NGPS and control iPSCs.

(a) BANF1c.34G>A mutation verified by DNA sequencing of NGPS-iPSCs. (b) RT–PCR analysis of pluripotency markers in representative clones from control and NGPS-iPSCs. RNA from human fibroblasts (HF) and hESC was used as a negative and positive control, respectively. (c) Representative series of hematoxylin-eosin (H&E) stained sections from teratomas produced from control, N-I and N-II-iPSCs are shown. All of them formed teratomas with tissues representing all three embryonic germ layers. Bar, 100 μm. (d) DNA methylation profiling of control and NGPS fibroblasts and iPSCs. NGPS-iPSCs showed methylation patterns indistinguishable from control iPSCs, erasing all the alterations shown by progeroid fibroblasts. Unprocessed original scans of PCRs are shown in Supplementary Fig. 9.

Supplementary Figure 3 Gene Ontology analysis of epigenetic alterations in NGPS-fibroblasts.

Genes that showed significant differences in methylation status in NGPS fibroblasts as compared with controls were analysed through gene ontology for functional annotation.

Supplementary Figure 4 NF-κB regulation of somatic cell reprogramming.

(a) NGPS-fibroblasts were transduced either with pBABE-empty vector, IκBα-SR or IKK-kinase inactive (KI) and reprogrammed into iPSCs. Plot represents the number of Tra-1-60 positive colonies. Triplicates were done for each condition and mean values are represented. pBABE versus SR, P = 0.0001; pBABE versus IKK2-KI, P = 0.0002. (b) Control fibroblasts expressing a BANF1-specific shRNA were transduced either with pBABE, IκBα-SR or IKK2-KI and reprogrammed. Plot represents the number of Tra-1-60 positive colonies. n = 3, pBABE versus SR, P = 0.0001; pBABE versus IKK2-KI, P = 0.0001. (c) iPSCs derived from NF-κBi-treated cultures express pluripotency-associated markers in a similar extent to untreated iPSCs. RT–PCR results from representative clones are shown. RNA from human fibroblasts was used as negative control. (d) qRT–PCR of OCT-4 and NANOG in iPSCs derived in the presence of NF-κB inhibitors. n = 3 independent experiments. (e) Relative proliferative values of control-, IKK2-CA or Tax-transduced fibroblasts. (f) qRT–PCR of DOT1L (BANF1, P = 0.008; NGPS-edited, P = 0.006), YY1 (BANF1, P = 0.009; NGPS-edited, P = 0.007), NANOG (BANF1, P = 0.002; NGPS-edited, P = 0.003) and LIN28A (BANF1, P = 0.04; NGPS-edited, P = 0.03) in NGPS corrected fibroblasts. n = 3 independent experiments. (g) qRT–PCR of DOT1L in control and IKK2-CA (P = 0.005) or Tax-transduced fibroblasts is shown (P = 0.003). n = 3 independent experiments. For the experiments with iPSCs, at least three different clones were analysed for each genotype and representative results are shown. Error bars indicate SEM (P < 0.05; P < 0.01, two-tailed Student’s t test). Unprocessed original scans of PCRs are shown in Supplementary Fig. 9.

Supplementary Figure 5 Premature senescence in NGPS fibroblasts.

(a) Relative proliferation values of control, NGPS and Cas9-edited NGPS fibroblasts. (b) SA-β-gal staining in the same cells. Percentage of cells with positive staining is represented. n = 100 cells assessed in 3 independent fields (also hereafter in similar experiments). P = 0.0002. Bar, 200 μm. (c) Percentage of cells with positive nuclear staining of anti-BrdU antibody. P = 0.0002. (d) Relative proliferation values of control and NGPS fibroblasts transduced with pBABE or BANF1 are represented. CTR versus NGPS, P = 0.001; pBABE versus BANF1, P = 0.002. (e) SA-β-gal staining in the same cells. CTR versus NGPS, P = 0.002; pBABE versus BANF1, P = 0.002. Bar, 200 μm. (f) Percentage of cells with positive nuclear staining of anti-BrdU antibody. Error bars indicate SEM (P < 0.01, two-tailed Student’s t test).

Supplementary Figure 6 NF-κB regulation of p53-mediated senescence.

(a) Western-blot analyses of p53, p21 and p16 proteins in control and NGPS fibroblasts transduced with pBABE or IκBα-superrepressor. β-actin was used as a loading control. A representative image is shown of 3 independent experiments. (b) Senescence induction with hydrogen peroxide (0.1 mM for 2 h) in control fibroblasts transduced with pBABE or IκBα-SR. Percentages of SA-β-gal (P = 0.001) and anti-BrdU positive cells (P = 0.002) are depicted and representative pictures from SA-β-gal staining are shown. n = 100 cells assessed in 3 independent fields (also hereafter in similar experiments). Bar, 200 μm. (c) p16 western-blot analyses in hydrogen peroxide-treated control fibroblasts. β-actin was used as a loading control. (d) NGPS fibroblasts transduced with TP53 shRNA and IκBα-SR were reprogrammed into iPSCs. Plot represents the number of Tra-1-60 positive colonies. n = 3 independent experiments for each condition and mean values are represented. NGPS versus shp53, P = 0.005; shp53 versus SR, P = 0.004. (e) Specific down-regulation of TP53 in NGPS fibroblasts did not affect NF-κB activation. NF-κB electrophoretic mobility shift assay in control fibroblasts transduced with BANF1 shRNA in the presence or absence of TP53 shRNA. Oct-1 probe was used as an endogenous control. Error bars indicate SEM (P < 0.01, two-tailed Student’s t test). Unprocessed original scans of blots are shown in Supplementary Fig. 9.

Supplementary Figure 7 NGPS senescence-associated secretory phenotype.

(a) Heat map represents unsupervised hierarchical clustering of top-altered secreted protein levels in NGPS and control fibroblasts and MSCs (n = 2). Data are displayed as Log2-transformed expression signals. (b) Heat map represents alterations in proteins annotated as members of the senescence-associated secretory phenotype. Data are displayed as Log2-transformed expression signals.

Supplementary Figure 8 Upstream regulation and effectors of NF-κB reprogramming barrier.

(a) ATM blockade prevented NF-κB activation in NGPS fibroblasts. NF-κB EMSA in NGPS fibroblasts in the presence or absence of ATM inhibitor. Oct-1 probe was used as an endogenous control. (b) Phospho-ATM western-blot analysis in control fibroblasts transduced either with BANF1 shRNA or the empty vector. Right panel, NF-κB EMSA in control fibroblasts transduced with shBANF1 in the presence or absence of ATMi. (c,d) The expression of wild-type BANF1 prevented ATM and NF-κB activation in NGPS cells. (e) Western-blot analysis of RelA in murine fibroblasts from the indicated genotypes. β-actin was used as a loading control. Mean relative mRNA levels are represented. (f) Enrichment score plot shown was obtained from GSEA analysis of control fibroblasts and iPSC transcriptional profiles (P < 0.05). (g) Mice were treated with two different DOT1L inhibitors (epz-4777 and epz-5676) and, as both inhibitors achieved a similar biological effect, experimental data were pooled. Ki-67 immunohistochemistry of skin from 3-month-old wild-type, untreated and DOT1Li-treated Zmpste24-deficient mice. Bar, 30 μm. (h) Representative photographs of spleen and thymus from 3-month-old wild-type, untreated and DOT1Li-treated Zmpste24-deficient mice. (i,j) Prevention of inflammation-associated alterations in intestinal mucosa. Representative hematoxylin-eosin (H&E) staining micrographs are shown from wild-type, untreated and DOT1Li-treated Zmpste24-deficient mice. Mean mucosa thickness (P = 0.006) and villus (P = 0.005) length were quantified and represented. Bar, 100 μm. n = 4 independent animals for each condition. (k) Colony forming assay in BM-MSCs from untreated and DOT1Li-treated Zmpste24−/− mice. P = 0.005. n = 3-untreated and 4-DOT1Li treated (epz-4777, 2 mice and epz-5676, 2 mice) Zmpste24-deficient mice. Error bars indicate SEM. P < 0.01, two-tailed Student’s t test). Representative images are shown in western-blot analyses from 3 independent experiments. Unprocessed original scans of blots are shown in Supplementary Fig. 9.

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Soria-Valles, C., Osorio, F., Gutiérrez-Fernández, A. et al. NF-κB activation impairs somatic cell reprogramming in ageing. Nat Cell Biol 17, 1004–1013 (2015). https://doi.org/10.1038/ncb3207

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