High-quality genome sequence of extremotolerant tardigrade

R. varieornatus is an extremotolerant tardigrade species, which becomes almost completely dehydrated on desiccation (Fig. 1a,b) and withstands various physical extremes4. The genome sequence of R. varieornatus was determined by using a combination of the Sanger and Illumina technologies (Supplementary Table 1). To minimize microbial contamination we cleansed egg surfaces with diluted hypochlorite and before sampling the tardigrades were starved and treated with antibiotics for 2 days. After the removal of short scaffolds (<1 kb) and mitochondrial sequences, we obtained the assembly spanning 56.0 Mbp (301 scaffolds). Coverage analysis (160 × Illumina sequencing) revealed that 199 scaffolds (99.7% in span) had considerable coverage (>40), whereas 102 scaffolds had exceptionally low coverage (<1; Supplementary Fig. 1 and Supplementary Data 1). We considered these 102 scaffolds (153 kb in span) as derived from contaminating organisms and excluded them from our assembly. As a result, our final assembly spans 55.8 Mbp (199 scaffolds; N50=4.74 Mbp; N90=1.3 Mbp; Supplementary Table 2). The span is highly concordant with the genome size estimated by DNA staining in the tardigrade cells (∼55 Mbp; Supplementary Fig. 2), suggesting sufficiency of our assembly span and no significant inflation by contaminated organisms. We also constructed a full-length complementary DNA library from dehydrated tardigrades and determined paired-end sequences. BLAST search of these Expression Sequence Tag (EST) data against our genome assembly revealed 70,674 of 70,819 sequences (99.8%) were successfully mapped (E-value<1065). The completeness of our assembly was also supported by high coverage (95.6%) in essential eukaryotic genes assessed by Core Eukaryotic Genes Mapping Approach16 (Supplementary Table 2) and the very low duplication rate in Core Eukaryotic Genes Mapping Approach (1.13) indicated that our assembly was largely free from inflation by contaminating organisms. We generated gene models based on our messenger RNA-sequencing (RNA-seq) data for six states (two embryonic stages and four states of adults during dehydration and rehydration) and merged them with ab initio gene models, to produce the comprehensive gene set, containing 19,521 protein-coding genes. The genome of this species was highly compact and, correspondingly, the mean length of coding sequences (1,062 bp), exons (234 bp) and introns (402 bp) were fairly short and genes were densely distributed with short inter-coding sequence distances (mean 1,099 bp; Supplementary Table 3).

Figure 1: The extremotolerant tardigrade R. varieornatus and taxonomic origins of its gene repertoire. (a,b) Scanning electron microscopy images of the extremotolerant tardigrade, R. varieornatus, in the hydrated condition (a) and in the dehydrated state (b), which is resistant to various physical extremes. Scale bars, 100 μm. (c) Classification of the gene repertoire of R. varieornatus, according to their putative taxonomic origins and distribution of best-matched taxa in putative HGT genes. Full size image

No extensive HGT in R. varieornatus genome

To evaluate the significance of HGT in the tardigrade gene repertoire, we first performed BLAST search against the non-redundant database of National Centre for Biotechnology Information. Among the 19,521 tardigrade proteins, 10,957 proteins (56.1%) had similar proteins below the threshold (E-value≤10−5) used to estimate HGT in rotifers17. The vast majority exhibited the best similarity with metazoan proteins and were thus classified as metazoan origin (10,249 proteins; 52.5% of total proteins; Fig. 1c). We examined putative HGT based on HGT indices that were calculated by subtracting the best bit score of the metazoan hit from that of the non-metazoan hit in BLAST searches, as used in previous reports10,17. Only 234 proteins (1.2%) had HGT scores higher than the previously defined threshold (≥30)10,17 and were classified as putative HGT genes (Fig. 1c and Supplementary Data 2). Of 234 putative HGT genes, 226 genes were encoded in the scaffolds containing metazoan-origin genes and all 234 putative HGT genes were supported by substantial coverage of genomic reads (Supplementary Fig. 3 and Supplementary Data 2), suggesting that these putative HGT genes were encoded in the tardigrade genome rather than mis-incorporated minor contaminating sequences. In our evaluation of genome assembly, we excluded 102 scaffolds due to the extremely low coverage as a sign of possible contamination origins. To examine the impact of this exclusion on the estimated HGT proportion, we applied the same gene prediction on the excluded scaffolds and found 152 additional protein-coding genes. Of these 152 genes, 129 exhibited high HGT indices (≥30) and were classified as putative HGT genes. Even taking into account these genes, the proportion of putative HGT genes was still only 1.8% (Supplementary Table 2). In any case, the proportion of HGT in our genome was much lower than those reported for the UNC assembly of H. dujardini (17.5%)10. In addition to the HGT proportion, we also found a striking contrast in putative taxonomic origins of HGT genes. In the UNC assembly, most (>90%) of the putative HGT genes were presumed to be of bacterial origin. In contrast, more than half (65%) of the putative HGT genes have probable eukaryotic origins in our assembly, mainly fungal origin (Fig. 1c).

Our transcriptome analyses revealed that 138 of 234 putative HGT genes were certainly transcribed (fragments per kilobase of exon per million mapped fragments ≥5) and were considered as functional (Supplementary Data 2). These functional HGT genes included several tolerance-related genes, for example, catalases. Catalase is an antioxidant enzyme that decomposes hydrogen peroxide, which is hazardous to the organism, and antioxidant enzymes are presumed important to counteract oxidative stress during desiccation18. In our assembly, we found three catalases and one putative pseudo-gene. All of them had high HGT scores and contained an extra domain at the carboxy terminus compared with other metazoan catalases (Supplementary Fig. 4). This structure resembles those of bacterial clade II catalases. Catalases are classified into three sub-groups, termed clade I, II and III, and all other metazoan catalases are classified as clade III19. Phylogenetic analyses confirmed the classification of tardigrade catalases as clade II (Supplementary Fig. 5).

Expansion of stress-related genes in the tardigrade genome

Comparison of the gene repertoire with other metazoans revealed characteristic expansion of several stress-related gene families such as superoxide dismutases (SODs) and MRE11 (Supplementary Fig. 6 and Supplementary Data 3). Sixteen SODs were found in our assembly, whereas less than ten SODs are found in most metazoans. SOD is a detoxifying enzyme of superoxide radicals, a type of reactive oxygen species (ROS)18. As desiccation induces oxidative stress, expanded SODs could contribute to better tolerance against desiccation18. MRE11, another expanded gene family, plays important roles in repair processes of DNA double-strand breaks (DSBs)20. Four MRE11 genes were found in our assembly, whereas most animals possess only one copy. DNA in tardigrade cells undergo DSBs during long preservation in a dehydrated state and expanded MRE11 might be beneficial for efficient repairing damaged DNA. In the UNC assembly of H. dujardini, expansions by HGT were reported for several other DNA repair genes such as Ku, umuC, Ada and recA (Rad51)10. We observed no significant expansion or sign of HGT for those genes in R. varieornatus (Supplementary Data 3). Furthermore, all MRE11 genes in R. varieornatus were suggested to be of metazoan origin (Supplementary Data 2). Thus, the expansion of MRE11 was likely to be due to gene duplication events during evolution to this lineage, rather than acquisition from other non-metazoan organisms through HGT. We also detected the expansion of some other gene families, for example, guanylate cyclases (Supplementary Fig. 6). Their relation to tardigrade physiology is, however, currently elusive.

Selective loss of peroxisomal oxidative pathway

We also evaluated whether some metabolic pathways had been lost in our tardigrade genome. To assess this, we mapped genes found in model organisms but missing in our tardigrade genome to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways21. Statistical analysis revealed the significant gene loss in the peroxisomal pathway (corrected P-value=0.007, Fisher’s exact test; Supplementary Data 4). Many oxidative enzymes including those in the conserved β-oxidation pathway and several peroxisome biogenesis factors were missing (Supplementary Figs 7 and 8). β-Oxidation is a major catabolic pathway of fatty acids, normally catalysed by two sets of enzymes, one in the mitochondria and the other in the peroxisome22. All members of the peroxisomal set were missing, whereas a complete set of mitochondrial enzymes was present (Supplementary Data 5), suggesting actual gene loss in the peroxisomal β-oxidation process rather than insufficient genome sequencing.

Selective loss of stress responsive pathways

In addition to the KEGG pathways, we searched for the non-curated gene networks lost in the tardigrade genome by connecting putative lost genes using the protein–protein interaction database, STRING23. We found that eight lost genes had an interconnected network in the highly conserved stress-responsive signalling pathways (Fig. 2 and Supplementary Data 6). Three of these genes, HIF1A, PHD and VHL, are central components to regulate response to hypoxia24. REDD1 is a downstream target of HIF1A25, as well as a downstream target induced by p53 on genotoxic stress26. REDD1 activates the TSC1/TSC2 complex, leading to downregulation of mammalian target of rapamycin complex 1 (mTORC1) activity25. The other lost gene, Sestrin, is also a downstream gene of p53 connecting genotoxic stress to mTOR signalling27. As TSC1/TSC2 is activated by oxidative stress28, the tardigrade lacks the signalling components connecting various stresses such as hypoxia, genotoxic stress and oxidative stress, to downregulation of mTORC1. In contrast, all other signalling components are present for regulation of mTORC1, depending on physiologic demands such as energy deprivation sensing29 and amino acid sensing30.

Figure 2: Selective loss of stress responsive signalling to mTORC1 downregulation. Gene networks involved in the regulation of mTORC1 activity. Magenta indicates genes absent in the tardigrade genome and green indicates retained genes. The interconnected eight genes mediating environmental stress stimuli to downregulate mTORC1 were selectively lost, whereas all components involved in sensing and mediating physiologic demands were present. Full size image

Constitutive abundant expression of tardigrade-unique genes

We examined gene expression profiles during dehydration and rehydration using mRNA sequencing and comparative analyses detected only minor differences (Supplementary Data 2), suggesting that the tardigrade can enter a dehydrated state without significant transcriptional regulation. This finding is consistent with the fact that this tardigrade, R. varieornatus, tolerates rapid desiccation by direct exposure to low humidity conditions. We speculated that putative protective proteins are constitutively expressed. During inspection of abundantly expressed genes, we noticed that many abundantly expressed genes are classified as tardigrade-unique genes that exhibited no or low similarity to non-tardigrade proteins (Supplementary Fig. 9).

These abundantly expressed proteins included previously identified tardigrade-unique heat-soluble proteins, CAHS and SAHS, both of which maintain solubility even after heat treatment and are proposed to be involved in the protection of biomolecules during desiccation31,32. We found significant expansion of these tardigrade-unique protein families, as 16 CAHS genes and 13 SAHS genes in our assembly, whereas no counterparts were found in other phyla, except 3 SAHS genes with low similarity to several metazoan fatty acid-binding proteins. In accordance with the identification of CAHS and SAHS proteins as predominant proteins in the heat-soluble proteome of the tardigrade, our transcriptome data confirmed the abundant expression of these family members in the adult stage as well as in embryonic stages, although dominantly expressed members differed depending on the stage (Supplementary Data 2). We found a reasonable number of genes unique to the species or the phylum (8,023 genes; 41.1% of the gene repertoire; Fig. 1c). Abundantly expressed unique genes might be good candidates involved in the tolerability of the tardigrade.

Identification of a tardigrade-unique DNA-associated protein

R. varieornatus exhibits extraordinary tolerance against high-dose radiation4. Considering DNA as a major target of radiation damage, we hypothesized that tardigrade-unique proteins associate with DNA to protect and/or to effectively repair DNA in the tardigrade. To explore this possibility, we isolated the chromatin fraction from the tardigrade and used tandem mass spectrometry to identify the proteins contained in the bands selective to the chromatin fraction (Supplementary Fig. 10). Among the identified proteins (Supplementary Tables 4 and 5), we examined subcellular localization of putative nuclear proteins by expressing them as green fluorescent protein (GFP)-fused proteins in Drosophila Schneider 2 (S2) cells. Only one protein, termed Damage suppressor (Dsup), co-localized with nuclear DNA (Supplementary Fig. 11) and similar co-localization was also observed in human cultured HEK 293T cells (Fig. 3a). Our transcriptome data revealed abundant expression of Dsup in an early embryonic stage (within the top 100 abundantly expressed genes; Supplementary Data 2), which is consistent because nuclear DNA extensively replicates in the embryonic stage. To verify the localization of Dsup protein in tardigrade cells, we performed immunohistochemistry with frozen sections of tardigrade embryos. In almost all tardigrade cells expressing Dsup, Dsup proteins co-localized with nuclear DNA (Supplementary Fig. 12).

Figure 3: Co-localization with nuclear DNA and mobility shift of DNA by Dsup. (a) Subcellular localization of Dsup-GFP fusion proteins transiently expressed in HEK293T cells. Nuclear DNA was visualized by Hoechst 33342. Scale bars, 10 μm. (b) Mobility shift of DNA by bacterially expressed Dsup protein in a dose-dependent manner (10, 50, 75 or 100 ng). Black arrowhead indicates the predicted size of the probe DNA (3 kbp, 10 ng). Red arrowhead indicates the position of the extremely slowly migrating DNA in the presence of Dsup protein. A similar extensive mobility shift was observed with histone H1. Full size image

Dsup protein showed no sequence similarity to any proteins or motifs in BLASTP and InterProScan searches. In silico prediction revealed a putative long α-helical region in the middle and a putative nuclear localization signal at the C terminus (Supplementary Fig. 13). Dsup protein is highly basic (pI=10.55), especially in the C-terminal region, suggesting its potential association with DNA through electrostatic interactions. Mutational analyses using variously truncated Dsup proteins fused with GFP revealed that the C-terminal region (Dsup-C) is required and sufficient for co-localization with nuclear DNA (Supplementary Fig. 14a–c). Expression of Dsup-C induced an abnormally aggregated distribution of nuclear DNA, whereas full-length Dsup-expressing cells had an almost normal distribution of nuclear DNA, similar to that in control cells (Supplementary Figs 14a and 15).

To examine the affinity of Dsup protein to DNA, we performed a gel-shift assay using bacterially expressed Dsup protein in vitro. Pre-incubation with purified Dsup protein significantly retarded the migration of linearized plasmid DNA in a dose-dependent manner (Fig. 3b), suggesting that Dsup protein has certain affinity to DNA in vitro. When Dsup protein was mixed with DNA at a 10:1 (wt:wt) ratio, the migration of DNA was almost completely inhibited. This retarded mobility of DNA could be due to formation of huge DNA–Dsup protein complexes and/or neutralization of the negative charge of DNA. These results suggested the physical affinity of Dsup protein to DNA molecules, although physiological specificity and mode of interaction between Dsup protein and DNA remain elusive. A similar drastic band shift was observed with the ubiquitous chromatin protein histone H1 (ref. 33). Dsup protein required the higher protein:DNA ratio for a complete band shift compared with histone H1, suggesting relatively weak affinity to DNA of Dsup than histone H1. Dsup protein lacking the C-terminal region (DsupΔC) completely lost the ability to shift the DNA mobility and Dsup-C alone was sufficient to shift the DNA band (Supplementary Fig. 14d). These findings indicated that the C-terminal region of Dsup is responsible for association with DNA as well as for co-localization with nuclear DNA.

Dsup protein suppresses DNA damage in human cultured cells

We hypothesized that the association of Dsup proteins with nuclear DNA might help to protect DNA from irradiation stress. To examine this possibility, we established a HEK293 cell line stably expressing Dsup under the control of the constitutive CAG promoter. Co-localization of Dsup protein with nuclear DNA was confirmed by immunocytochemistry in the established line (Supplementary Fig. 16a). X-ray irradiation induces various types of DNA damage, including DNA breaks, mainly single-strand breaks (SSBs). To examine the effect of Dsup on X-ray-induced DNA breaks, Dsup-expressing cells and untransfected HEK293 cells were exposed to 10 Gy X-ray irradiation. After irradiation, the cells were exposed to an alkaline condition (pH>13) to denature the damaged DNA and dissociated single-strand DNA fragments were analysed in single-cell electrophoresis (alkaline comet assay). The short fragmented DNA migrated to more distant location from the nuclei (comet tail region) and thus the proportion of DNA in the comet tail was considered an indicator of DNA breaks. In irradiated Dsup-expressing cells, the proportion of tail DNA was only 16%—less than half of that in the untransfected HEK293 cells (33%; Fig. 4a). This finding suggested that Dsup protein suppressed X-ray-induced SSBs in human cultured cells. There are two modes for X-ray to induce SSBs: the direct absorption of X-ray energy into the DNA (direct effects) and through attack by ROS generated from water molecules activated by X-ray energy (indirect effects)34. We, therefore, examined the effect of Dsup protein on DNA SSBs generation by ROS. Exposure to hydrogen peroxide induced severe fragmentation of DNA (71% of total DNA in tail) in control HEK293 cells. In contrast, DNA fragmentation in Dsup-expressing cells was substantially suppressed to only 18% of total DNA in the tail (Fig. 4b), indicating that Dsup protein was able to protect DNA from ROS as well as X-rays. Pretreatment with the antioxidant, N-acetyl-L-cysteine (NAC) also substantially suppressed peroxide-induced SSBs. The combination of NAC and Dsup led to even greater suppression, although the suppression induced by their combination was less than the sum of those in each condition individually, suggesting that NAC and Dsup at least partially share the same suppression mechanism, most probably counteracting oxidative stress.

Figure 4: Dsup protein suppresses stress-induced DNA fragmentation in human cultured cells. (a) The effects of Dsup on SSBs by 10 Gy X-ray irradiation in alkaline comet assays. The irradiated cells were immediately subjected to the assay. Representative images are shown for each condition. In the pseudo-coloured images in the inset, red to blue circles indicate nuclear DNA and magenta indicates fragmented DNA in tail. DNA fragmentation was assessed by the proportion of DNA detected in the tail region (% of DNA in Comet Tail). At least 281 comets were analysed for each condition. **P<0.01 and ***P<0.001 (Welch’s t-test: non-irradiated, t-value=−3.199, P-value=0.0015; irradiated: t-value=8.599, P-value<1.0E−15). (b) The effects of Dsup on SSBs caused by hydrogen peroxide (H 2 O 2 ) treatment in alkaline comet assays. Cells were treated with 100 μM H 2 O 2 for 30 min at 4 °C, to induce DNA damage with or without pretreatment with 10 mM NAC as an antioxidant for 30 min. At least 203 comets were analysed for each condition. ***P<0.001 (Tukey–Kramer’s test). (c) The effects of Dsup on DSBs by 5 Gy X-ray irradiation in neutral comet assays. Three hundred comets were analysed for each condition. **P<0.01 and ***P<0.001 (Welch’s t-test: non-irradiated, t-value=2.758, P-value=0.0060; irradiated; t-value=7.406, P-value=4.7E−13). Values represent mean±s.d. in all panels. Scale bars, 100 μm. Full size image

Besides SSBs, high-dose X-ray irradiation also induces DSBs, which are much more hazardous for organisms due to their difficult repair. We next examined the effect of Dsup protein on DSBs using a neutral comet assay, in which DNA fragmentation was analysed without dissociating in a neutral condition. The proportion of fragmented DNA was ∼40% reduced in Dsup-expressing cells compared with that in the untransfected cells (Fig. 4c). These findings together suggest that Dsup protein suppressed X-ray-induced DNA DSBs and SSBs.

We further verified the suppression of DNA breaks by Dsup proteins using another DSB quantification method. In irradiated cells, histone H2AX around DSBs becomes phosphorylated within an hour35, referred to as γ-H2AX, and γ-H2AX can be used as an indicator of DSBs. We visualized γ-H2AX by immunofluorescence and counted the number of foci per nucleus at 1 h after irradiation. For this experiment, we irradiated cells with a relatively lower dose (1 Gy) of X-ray to avoid overlap of neighbouring foci and minimize counting errors. The Dsup-expressing cells exhibited an ∼40% reduced number of γ-H2AX foci compared with untransfected cells (Fig. 5a). We further established Dsup knockdown cells by transfecting a small hairpin RNA (shRNA) expression construct in Dsup-expressing cells. Dsup expression was successfully reduced by 77% in the knockdown cells (Fig. 5b). The reduction of DNA damage completely disappeared by Dsup knockdown (Fig. 5c,d). These findings indicated that Dsup protein is responsible for suppressing DNA damage in irradiated human cultured cells. When using a stable line expressing mutant Dsup protein, DsupΔC, which lacks the C-terminal DNA-associating region (Supplementary Fig. 16b), we detected no reduction of DNA fragmentation in the alkaline comet assays (Supplementary Fig. 17a), suggesting that the association with DNA is prerequisite for Dsup protein to protect DNA from X-ray. This view was further supported by the impaired suppression of the γ-H2AX foci in the DsupΔC-expressing cells (Supplementary Fig. 17b).

Figure 5: Reduced formation of γ-H2AX foci in human cultured cells depending on Dsup expression. (a) Distribution of the numbers of γ-H2AX foci per nucleus is shown. Each dot represents an individual nucleus of a HEK293 cell (Control) or a Dsup-expressing cell (Dsup) under non-irradiated and irradiated conditions. ***P<0.001; NS, not significant (Welch’s t-test). (b) Significant decrease of Dsup transcript in shRNA-introduced cells (Dsup+shDsup) compared with that in untreated Dsup-expressing cells (Dsup shRNA(−)). n=3. Values represent mean±s.e.m. ***P<0.001 (Student’s t-test). (c) Quantitative comparison of γ-H2AX foci number among untransfected HEK293 cells (Control), Dsup-expressing cells (Dsup) and Dsup-knockdown cells (Dsup+shDsup) under non-irradiated and 1 Gy X-ray irradiated conditions. At least 70 cells were analysed for each condition. Values represent mean±s.d. **P<0.01; NS indicates not significant (Tukey–Kramer’s test). (d) Representative images detecting γ-H2AX foci in each condition. Fluorescent images were converted to binary images for automatic counting of foci. Scale bar, 10 μm. Full size image

Dsup improves viability of irradiated human cultured cells

To test whether DNA protection by Dsup protein could also improve cellular survival after irradiation, we measured the cell viability after irradiation. In general, 3–7 Gy of X-ray induces severe DNA damage in mammalian cells, leading to loss of proliferative ability36. Accordingly, we irradiated cells with 4 Gy X-ray at 1 day post seeding (dps), which was the minimum dose enough to suppress proliferation of untransfected HEK293 cells in our condition. After irradiation, cell proliferation was examined at 24 h intervals for 8 days using PrestoBlue Cell Viability reagent, which measures the total reducing power of the cell culture37. Dsup-expressing cells exhibited slightly better cell viability after irradiation compared with those of untransfected HEK293 cells (Supplementary Fig. 18a–c). At 4 days after the cell viability analysis (12 dps), we noticed a drastic difference between Dsup-expressing cells and untransfected cells under phase-contrast microscopy (Supplementary Fig. 18d). Almost all irradiated untransfected cells had an abnormal round shape and were mostly detached from the culture dish, typical characteristics of dead cells. In contrast, many irradiated Dsup-expressing cells had a normal morphology and attached to the culture dishes, suggesting that these cells retained the characteristics of live adherent cells and perhaps even had proliferative ability.

To confirm their proliferative ability, we examined the temporal change in cell numbers over a longer period after irradiation with 4 Gy of X-ray. Even under non-irradiated conditions, Dsup-expressing cells proliferated slightly faster than the untransfected cells, whereas Dsup-knockdown cells exhibited similar proliferation to that of untransfected cells (Fig. 6b). At 10–12 dps, the cell numbers became nearly saturated. Under irradiated conditions, almost all untransfected cells detached from the culture dish and had an abnormal round shape (Fig. 6a). In contrast, some of the irradiated Dsup-expressing cells attached to the culture dish with an apparently normal morphology and such cells increased over time (Fig. 6a). Cell counting analyses confirmed these observations. At 8 dps, the number of irradiated untransfected cells was almost unchanged from that at the seeding and further decreased at 10 and 12 dps (Fig. 6b). In contrast, the number of Dsup-expressing cells increased even at 8 dps compared with that at the seeding and drastically increased at 10 and 12 dps (Fig. 6b), suggesting that at least some fraction of irradiated Dsup-expressing cells retained proliferative ability. Growth rates at 8–12 dps were comparable to those of non-irradiated Dsup-expressing cells. In Dsup-knockdown cells, the improvements in cell viability and proliferative ability were completely abolished and their phenotypes were similar to those of untransfected HEK293 cells (Fig. 6). These findings suggested that Dsup protein confers increased radiotolerance to human cultured cells. Cells expressing a Dsup mutant lacking the DNA-associating domain (DsupΔC) exhibited impaired improvement of radiotolerance compared with those expressing full-length Dsup protein (Supplementary Fig. 19), suggesting that DNA targeting is important for full improvement of the radiotolerance by Dsup. As radiosensitivity of mammalian cells is affected by the cell cycle38, we compared the cell cycle distribution between Dsup-expressing cells and untransfected cells using flow cytometry. However, no significant differences were detected (Supplementary Fig. 20), suggesting that the improved radiotolerance conferred by Dsup protein was not due to alterations of the cell cycle.