Transformation of naked mole-rat cells

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Fig. 1: NMR cells can be transformed by SV40LT and HRASG12V.
Fig. 2: NMR transformation is not dependent on the promoter, vector or culturing medium used.

Data availability

The RNA-sequencing data are available from the European Nucleotide Archive (accession number E-MTAB-8932). Raw western blot data are provided as Supplementary Fig. 1. The authors declare that all remaining supporting data are available within the paper and the Supplementary Information or from the corresponding authors upon reasonable request.

Code availability

The source code used for analysis of the RNA-sequencing data is available at https://github.com/KaBach/NMR.

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Acknowledgements

We thank M. Waterhouse for reading of and comments on the manuscript, S. Nik-Zainal for access to EVOS FLA2 and the staff at the Sanger Institute, Research Service Facility (RSF) for their assistance. F.H. is funded by a Gates Cambridge Trust PhD scholarship. Y.K. is funded by a CRUK multidisciplinary award to E.S.J.S. K.A.L. is funded by a CRUK career establishment award and The Isaac Newton Trust Grant to W.T.K. K.B. is funded by CRUK Cambridge Centre PhD studentship. R.U. is funded by NC3Rs PhD studentship. This work was funded by donations from P.B., Magdalene College (Cambridge), The Isaac Newton Trust Grant (16.38c) and a CRUK Grant (C56829/A22053) to E.S.J.S. and a CRUK grant (C47525/A17348) to W.T.K.

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Authors

Contributions

F.H. designed and carried out most of the experiments and analysed data. Y.K. helped with cell line generation and performed western blots. K.A.L. and R.U. helped with xenograft experiments. K.B. analysed the RNA-sequencing data. P.B., E.S.J.S. and W.T.K. conceptualized the original ideas for the project. E.S.J.S. and W.T.K. conceived and supervised the study. F.H., E.S.J.S. and W.T.K. wrote manuscript with input from all other authors.

Corresponding authors

Correspondence to Ewan St John Smith or Walid T. Khaled.

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The authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 NMR cells from different tissues can be transformed.

a, Schematic of the lentiviral vectors generated and used in this study. CMV, cytomegalovirus promoter; RU5, 5ʹ long-terminal repeat lacking the U3 region; hU6, human U6 promoter;iScaffold, improved guide RNA (gRNA) scaffold; PGK, mouse Pgk1 promoter; EF1α, human EF1A promoter; SV40E, SV40 early region promoter; CMVIE, CMV immediate early promoter; Puro, puromycin resistance gene; 2A, Thosea asigna self-cleaving 2A peptide; ΔU3RU5, enhancer-deleted 3ʹ long-terminal repeat. b, Schematic representation of the different cell lines generated as part of this study using the vectors shown in a. cf, Quantification of soft-agar assay colonies from different cell lines generated from NMR cells from the pancreas (c), lung (d), kidney (e) and intestine (f). For each organ, cells were derived from four different NMRs, except in the case of intestine, for which cells were derived from two different NMRs. Different shapes represent different experimental repeats. Each experiment was performed up to four times and each experimental repeat included six technical replicates. Each data point represents the number of colonies observed from an individual technical replicate. In total, more than 3,300 fields of view (non-overlapping images) were analysed for the pancreas, whereas more than 3,200, 3,800 and 1,300 fields of view were analysed for the lung, kidney and intestine, respectively. It is worth noting that kidney cells can be transformed with SV40LT alone as shown in e. cf, Data were analysed using Wilcoxon rank-sum tests; ***P ≤ 0.0001; NS, not significant. Box plots are shown as follows: centre line, median; box limits, upper and lower quartiles; whiskers, 1.5× the interquartile range.

Extended Data Fig. 2 Transformed NMR cells form tumours in NSG mice.

a, Quantification of xenograft tumour growth in NSG mice injected with primary or Pgk1-SV40LT;HRASG12V-transduced kidney and lung cells. Each cell line was injected into four mice; each mouse is represented by a single line. Colours represent different vectors and shapes represent different tissues. b, Representative images of xenograft tumours shown in Fig. 1b. c, Quantification of xenograft tumour growth in NSG mice injected with primary or Pgk1-SV40LT;HRASG12V-transduced NMR skin cells obtained from Tian et al.5. Each cell line was injected into four mice; each mouse is represented by a single line. d, Representative images of xenograft tumours reported in Fig. 1d. e, Western blots showing the expression of SV40LT and HRASG12V from different promoters in NMR skin cell lines. f, Quantification of soft-agar colonies from NMR skin cell lines generated by introduction of Pgk1-SV40LT or Pgk1-SV40LT;HRASG12V through lentiviral particles. In each set of experiments, lentiviral particles used for introducing Pgk1-SV40LT;HRASG12V were titrated to keep the number of lentiviral particles transducing each cell to around 1. Each experiment was repeated up to five times (represented by different shapes) and each experimental repeat included six technical replicates. Each data point represents the number of colonies observed from a single technical replicate. Data were analysed using Wilcoxon rank-sum tests; ***P ≤ 0.0001; NS, not significant. Box plots are shown as follows: centre line, median; box limits, upper and lower quartiles; whiskers, 1.5× the interquartile range.

Extended Data Fig. 3 Gene expression analysis of transformed NMR cells.

a, Loadings of PC2 for all genes used to compute the PCA in Fig. 2f ordered by the loading. The top ten genes with the highest or lowest loadings are highlighted in blue. Gene-set enrichment was performed on all genes with a loading higher or lower than 0.02 or −0.02, respectively. The top 5 Gene Ontology (GO) terms (biological processes) are visualized in the plot. Positive PC2 values were associated with untransformed cells whereas negative PC2 values were associated with transformed cells. b, Results of the differential expression analysis of various NMR cell lines. Left, transformed cells generated in this study (Hadi et al.) were compared with transformed cells from Tian et al.5. Right, transformed cells generated in this study were compared with untransformed from this study. The top 20 differentially expressed (DE) genes as well as the transgenes present in the samples are highlighted in blue. The dashed line represents an FDR-adjusted threshold of P = 0.01. The following transgenes are highlighted in the volcano plots: Puro, SV40LT and RASG12V, which encode PuroR, SV40LT and HRASG12V, respectively, in the Pgk1-SV40LT;HRASG12V vector generated in the current study (Extended Data Fig. 1a); largeT and RasV12 encode SV40 large T antigen from pSG5-largeT (Addgene, 9053) and HRASG12V from pCMV-RasV12 (Clontech, 631924), respectively, and are from Tian et al.5.

Supplementary information

Supplementary Information

This file contains the Supplementary Methods.

Reporting Summary

Supplementary Information

This file contains the uncropped western blot images for Extended Data Figure 2e (Supplementary Figure 1), a list of primers used in the study (Supplementary Table 1) and a list of antibodies used in the study (Supplementary Table 2).

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Hadi, F., Kulaberoglu, Y., Lazarus, K.A. et al. Transformation of naked mole-rat cells. Nature 583, E1–E7 (2020). https://doi.org/10.1038/s41586-020-2410-x

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