Characterization of Common Minke Whale (Balaenoptera Acutorostrata) Cell Lines Immortalized with the Expression of Cell Cycle Regulators

Primary cultured cells cannot proliferate infinite. The overcoming of this limit can be classified as immortalization. Bypass of p16 senescence protein induces efficient immortalization various types of mammalians is previously reported. However, the Cetacea species is not known. Here, that common minke whale‐derived cells can be immortalized with a combination of human genes, mutant cyclin‐dependent kinase 4 (CDK4R24C), cyclin D1, and Telomerase Reverse Transcriptase (TERT) is reported. These results indicate that the function of cell cycle regulators in premature senescence is evolutionarily conserved. This study describes the conserved roles of cell cycle regulators in the immortalization of cells from humans to Cetacea species. Furthermore, using RNA‐seq based on next‐generation sequencing, the gene expression profiles of immortalized cells are compared with parental cells as well as those immortalized with SV40 large T antigen, which is once a popular method for cellular immortalization. The profiling results show that newly established common minke‐whale‐derived immortaliozed cells have completely different profiles from SV40 cells. This result indicates that the expression of mutant CDK4, cyclin D1, and TERT enables to establish immortalized cell lines with different biological nature from SV40 expressing cells.


Introduction
Based on the genetic analysis of the retrotransposon, Cetacea was evolutionarily developed from Artiodactyla. [1]Cetartiodactyla is a new classification of animals, derived from Cetacea and Artiodactyla. [2] similarity comparison of short interspersed elements (SINE) among multiple species revealed a complex relationship between Cetacea and Artiodactyla.[3] Large body size is a biological characteristic of Cetacea.Animals with large body size and long-life span require more cell division until maturation and they might be at a high risk for cancer development since the error rate of DNA replication would be relatively consistent.No relationship between cancer risk, body size, and longevity has been reported.[4] This finding is called "Peto's Paradox". [5]s one of the answers to this paradox, amplified p53 related tumor suppressor genes and DNA damageresponsive genes were reported in the elephant genome.[6] In line with this notion, we previously stated that African Savanna elephant-derived cells are difficult to immortalize with mutant cyclin dependent kinase 4 (CDK4), cyclin D1, and telomere reverse transcriptase (TERT); hereafter, we will refer to the immortalization method as the K4DT method in this manuscript.[7] We hypothesized that if the lifespan of elephant-derived cells is mainly regulated by the tumor suppressor p53 signal, unsuccessful immortalization of elephant-derived cells using the K4DT method might be due to p53 hyperactivation, as aberrant inactivation of the p16/pRB pathway was reported to activate p53.The K4DT process can bypass premature senescence induced by the activation of the retinoblastoma tumor suppressor protein, pRB.[8] The failure of the K4DT method on African Savana elephants motivated us to think about the immortalization of Cetacea-derived cells, as it has been postulated that TP53 gene amplification occurred specifically in elephant lineage species such as elephants and mammoths, but not in whale lineage species.[9] We tried to determine if the life span of the common minke whale (Balaenoptera acutorostrata)-derived fibroblasts are mainly restricted by p53 as in African savanna-elephant-derived fibroblasts.
Primary cells cannot continue proliferation infinitely. [10,11]The limitations of cell proliferation are classified into two categories.First, cell culture stress, primary cells are continuously subjected to cell culture stress as the ideal condition for cell culture varies depending on the cell type.For example, we previously reported that megabat-derived parental cells halted cell proliferation in early passages, which may be explained by cell culture stress. [12]econd, Hayflick's limit, a condition where the telomere repeat sequence at the end of the chromosomes is shortened leading cultured cells to halt cell proliferation. [13]Immortalization, characterized as infinite cell proliferation, is the technique to overcome these limitations. [10]he expression of oncogenic virus-derived proteins, such as SV40 large T [14] or human papilloma virus-derived E6E7 oncoprotein [15] is effective for immortalization.However, due to the loss of function of the p53 protein, which is essential to maintaining the intact chromosome condition, chromosomal abnormalities are frequently observed as genomic instability. [15,16]The critical role of the p53 tumor suppressor can be characterized as gradian of the genome. [17]As an alternative method of immortalization, combinational expression of R24C mutant type cyclin-dependent kinase 4 (CDK4) and cyclin D1, and telomerase reverse transcriptase (TERT) has been reported in human myoblasts. [18]It should be noted that the combined expression of mutant CDK4, cyclin D1, and TERT enabled infinite cell proliferation without genomic instability, which may explain the intact function of the p53 tumor suppressor. [16,19]f these genes, CDK4, cyclin D1, and TERT, was named as "K4DT" method. [18]Furthermore, we demonstrated that the K4DT method could be applied to immortalize cells derived from multiple species, including human, [20] bovine, [21] swine, [22] monkey, [23] midget buffalo, [24] wild cat, [25] prairie vole, [26] rat, [27]  and sea turtle. [28]The conserved amino acid sequence and basic mechanism of cell cycle regulators are the basis for cellular immortalization among multiple species.These results led us to introduce human-derived mutant CDK4, cyclin D1, and TERT into primary cells of common minke whales.This report aims to establish immortalized cells from Cetacea with human-derived cell cycle-regulating genes.Furthermore, the establishment of immortalized common mink whale derived cell might be able to reduce the number of sacrificed whales even for the research purpose.

Immortalization of Common Minke Whale Cells
We hypothesize that the poor proliferation potential of parental cells could be due to culture stress, but not due to telomere shortening.We first evaluated the efficiency of the gene introduction using a recombinant lentivirus expressing the Kusabira orange (KO) fluorescence protein.As shown in Figure 1A, an intense red fluorescence was observed in ≈30% of the cells.Since cellular immortalization with mutant CDK4, cyclin D1, and TERT requires simultaneous infection with these three genes, it is essential to estimate the efficiency of genetic introduction.We confirmed that the titers of the lentiviruses, CSII-CMV-CDK4R24C (2.03 × 10 6 cfu mL −1 ), CSII-CMV-cyclin D1 (2.02 × 10 6 cfu mL −1 ), CSII-CMV-TERT (2.00 × 10 6 cfu mL −1 ) were very similar to that of CSII-CMV-Kusabira Orange (2.02 × 10 6 cfu mL −1 ) and simultaneously inoculated these lentiviruses into Moo2 and Moo3 primary cells.The cells transduced with K4DT (CDK4R24C, Cyclin D1, and TERT) as well as those with K4D (CDK4R24C and Cyclin D1), showed extended life spans compared with Moo2 and Moo3 parental cells (Figure 1A).To address the efficiency of gene introduction, we mixed lentiviruses which express EGFP and KO, CSII-CMV-EGFP and CSII-CMV-KO, then exposed to the parental cell of Moo2 (Figure 2).As the results, we obtained EGFP expressing lentivirus was introduced into 98.1% of parental cell.In case of KO, it was 97.1%.From these data, we concluded the multiple infections with mixed lentivirus can be archived under our experimental condition.The morphology of K4D and K4DT cells was similar to that of the parental cells (Figure 1B).Transgene integration into cells was confirmed by polymerase chain reaction (Figure 2A; Figures   S1 and S2, Supporting Information).Next, we detected the expression of mutant CDK4 and cyclin D1 at the protein level using western blotting (Figure 2B; Figures S3 and S4, Supporting Information).

Cell Cycle Analysis of Recombinant Cells
Next, we evaluated the cell cycle of the parental and recombinant cells.We obtained data from the passage 1 of primary cells and passage 6-7 of K4D and K4DT cells.As shown in Figure 3A, K4D and K4DT cells presented an almost identical histogram in the flow cytometry analysis.We also evaluated the ratio of G0/G1, S phase, and G2/M phases in six replicates.K4D and K4DT cells showed significantly lower G0/G1 and increased G2/M ratios, suggesting that cell cycle turnover is accelerated in these cells (Figure 3B).

Sequential Passage of Recombinant Cells
We evaluated the growth potential of parental and recombinant common minke whale cells in sequential passages.As shown in Figure 4A, parental Moo2 and Moo3 halted cell pro-  liferation at passage 2. Although a partial portion of parental cells showed positive staining for senescence-associated betagalactosidase staining (SA--gal staining), most of them were negative for staining activity, indicating that parental cells halted cell proliferation based on cell culture stress, not due to the shortening of the telomere sequence (Figure 4B, left panels).While K4DT cells continued to proliferate after reaching a PD value of 100, K4D cells showed proliferation around the PD value of 20-50.When K4D cells stopped proliferating they stained positively for SA--gal, as shown in Figure 4B (middle panels) and C (left panel), while K4DT cells were free from positive staining for SA--gal, as shown in Figure 4B,C (right panels).Based on these observations, we concluded that we obtained newly established cell lines from common minke-whale-derived muscle.

Preparation of SV40 Expressing Cell and Whole Gene Transcriptome Analysis
The establishment of cell lines derived from whale-related species has been achieved mainly by the expression of the SV40 large T antigen and TERT. [29]To compare the biological nature of our newly established common minke whale-derived K4DT cells, we also showed immortalized cells from Moo2 with SV40T antigen by retrovirus-mediated gene transfer.To estimate the infection efficiency of SV40T antigen expressing retrovirus, we used EGFP expressing retrovirus as the surrogate marker.After monitoring that the efficiency of retrovirus infection with enhanced green fluorescence protein (EGFP)-expressing retrovirus was ≈5-7% (Figure S5A, Supporting Information).Based on the infection efficiency of EGFP expressing retrovirus, we estimated that efficiency of SV40 gene delivery would be ≈5% of cell population.After infection, we observed the emergence of smaller cells (Figure S5B, Supporting Information).We waited around one month until the SV40 cell showed growth advantage in the cell culture dish.SV40 cells also showed an extended life span.

Whole Gene Transcriptome Analysis
We first mapped reads into the reference genome using STAR and the reference genome sequence of the common minke whale (https://www.ncbi.nlm.nih.gov/assembly/GCF_000493695.1/,Balaenoptera acutorostrata, BalAcu1.0).However, the downloaded reference information did not contain a gene reference gtf file.As the replacement, we used the Balaenoptera musculus genome (Blue whale, mBalMus1.pri.v3).Workflow of the transcriptome analysis is presented in Figure 5A.Sequencing data were obtained from three biological replicates.The quality of the RNA-seq reads is shown in Figures S6-S8 (Supporting Information).Gene expression data of all genes after the counting with FeatureCounts are listed in the database of Figshare (https://doi.org/10.6084/m9.figshare.22082975.v1.).The correlation plots of all obtained data are presented in Figure 5B.The parental, K4DT, and SV40 cells showed unique clusters, indicating that the sequencing data were quite reproducible.The number of total reads for each sample and the mapping ratio against the reference sequence is shown in Figure 5C.The mapping ratio    was ≈87-93%, which is similar to our previous data on humanderived cells. [7,30]To estimate the cell origin of parental primary cell, we carried out the comparison of whole transcriptome with RNA-Seq data of of brain(SRR918699), heart(SRR918701), lung(SRR922125), kidney(SRR919295), liver(SRR919296), muscle(SRR922171, SRR922446) of Balaenoptera acutorostrata, and fibroblasts of Balaenoptera musculus(SRR11045090).As the result of 3D principle component analysis (PCA), the expression profiling of parental cell has mapped into the most close position from fibroblasts of Balaenoptera musculus (Figure 5D).The expression profiling of brain, heart, lung, kidney, liver, and muscle were more distant from parental cell.From this data, we concluded that our obtained parental cell has close nature to the fibroblasts.The movie of the detailed feature of PCA was listed in Figshare (https://doi.org/10.6084/m9.figshare.22083002.v1).As the next step, we compared the expression profiling of parental cell, K4DT cell, and SV40 cell.As the result of PCA, parental cell, K4DT cell, SV40 cell showed unique clusters, indicating that biological nature of K4DT is much different from SV40 cell (Figure 5E).The result of PCA is shown in Figshare (https: //doi.org/10.6084/m9.figshare.22083020.v1).To ensure the gene expression quantitation, we first narrow down the genes that have at least 1000 counts in any sample.To elucidate the biological difference of K4DT and SV40 cell, we did the pairwise comparison between parental cell-K4DT cell, and parental cell-SV40 cell (Figure 5F).We extracted 803 K4DT specifically expressed genes (Figure 5F).Furthermore, we also extracted 734 SV40 specifically expressed genes.The commonly expressed 2276 genes were excluded from the characterization (Figure 5F).The expression pattern of K4DT specifically down-regulated genes are shown in Figures 6 and 7.The heatmap of K4DT specifically up-regulated genes are also listed in Figures 8 and 9.The expression pattern of SV40 specifically down-regulated genes are shown in Figures 10 and 11.The heatmap of K4DT specifically up-regulated genes are also listed in Figures 12-14.Based on the gene list of 803 K4DT specifically expressed genes and 734 SV40 specifically expressed genes, we did the pathway analysis.The list of 803 genes specific to K4DT (https://doi.org/10.6084/m9.figshare.22099550)and 734 genes for SV40 (https://doi.org/10.6084/m9.figshare.22099568.v1)are listed in Figshare.The list of the pathways listed as top 3 are summarized in Table1.[33] In K4DT cell, Choline metabolism in cancer, SNARE interactions in vesicular transport, Longevity regulating pathway -multiple species were listed.We marked the down regulated or up regulated genes in the map (Figures 15-17).While there is overlap of molecules in multiple pathways, genes that related to the cell growth and DNA replication were found.In SV40 cell, pathways of Endocytosis, Hippo signaling pathway, Human immunodeficiency virus 1 infection were listed (Table 1).We marked the down regulated or up regulated genes in the map (Figures 18-21).The up-regulation of Arf, which is important for the membrane trafficking, up-regulation of Smad2/3, which is important for Bone morphogenetic protein (BMP) and transforming growth factor beta (TGF-beta) signaling.Since the listed pathways were completely different between K4DT and SV40, we concluded that those two cell lines have much different biological nature.
Figure 13.Heatmap analysis of SV40 specifically up-regulated genes, part1.The red color is highly expressed genes, white is middle, and blue is low expressed genes.

Figure 14.
Heatmap analysis of SV40 specifically up-regulated genes, part2.The red color is highly expressed genes, white is middle, and blue is low expressed genes.

Figure 15.
Heatmap analysis of SV40 specifically up-regulated genes, part3.The red color is highly expressed genes, white is middle, and blue is low expressed genes.

Discussion
In this study, we established common minke-whale-derived immortalized cells.We detected genomic integration of humanderived transgenes and protein expression with lentivirusmediated gene transfer.Furthermore, we obtained the data that our established cell maintains cell proliferation above the population doubling value of 100, which is one of the general criteria of immortalized cells.They were negative for SA--gal staining.From these observations, we conclude that we successfully established new common minke whale-derived immortalized cells.
For immortalization, we used the human-derived R24C mutant forms of CDK4 (CDK4 R24C ), cyclin D1, and TERT.Our observations indicate that the basic mechanism for the cell cycle turnover is conserved between humans and Cetacea.We previously succeeded in immortalizing cells derived from various mammalian species.However, in the case of African savanna elephants, although they are mammals, the introduction of CDK4 R24C , cyclin D1, and TERT could not achieve immortalization. [7]Based on the genomic analysis of elephants, p53-related genes are primarily amplified and they play a critical role in preventing the risk of cancer. [6]Sulak et al., showed that genomic amplification of p53 is limited in elephant related species (African savannah elephant, Asian elephant, Columbian mammoth, Woolly mammoth) but not in the mink whale. [34]ulak et al., also showed that the elephant genome encodes 20 copies of the tumor suppressor gene TP53 and that the increase in TP53 copy number occurred coincident with the evolution of large body sizes, the evolution of extreme sensitivity to genotoxic stress, and a hyperactive TP53 signaling pathway in the elephant (Proboscidean) lineage. [34]In this study, we showed that the K4DT method could efficiently immortalize minke whale muscle-derived fibroblasts, which were also virtually immortalized with the SV40 large T antigen.Sea turtles can also be listed as long-life-span animals.In our previous study, dramatically enhanced proliferation of sea turtle-derived cells was obtained using the K4DT method, [8] although TERT activity was not detected.These results and our previous successful immortalization of cells from various mammalian species indicate that the K4DT method is useful to most mammalian cells.
Furthermore, we compared the whole gene expression profiles of parental, K4DT immortalized, and SV40 immortalized cells with next-generation sequencing in this study.The profiling results showed that common minke-whale-derived K4DT cells had completely different profiles from SV40 cells.This result indicates that the expression of mutant CDK4, cyclin D1, and TERT enables us to establish immortalized cell lines with different biological nature from SV40 expressing cells.
In the global expression analysis with PCA of this study, the distance from parental cell-K4DT cell was almost identical with that parental cell-SV40 cell.In our previous study of human dermal papilla cell, SV40 immortalization caused more larger effect to the whole transcriptome than that of K4DT. [16]We cannot explain exact reason for the inconsistent results between mink whale and human dermal papilla cell.However, there might be species specific response even against the virus derived oncogene product.Our established cells will contribute to the understanding of the biological features of Cetacea-related species.We hope that our established cells will contribute to the progress of whale biology and give us a clue to understanding the molecular mechanisms by which whales appear in the history of animal evolution.

Conclusion
In this study, we established the immortalized cells from common minke whale muscle derived primary cell through the expression of human derived mutant CDK4, cyclin D1, and TERT.Our observations indicate that function of those cell cycle regulators is evolutionally conserved from human to Cetacea.Our study would enable us to efficiently establish the cell lines derived from Cetacea related species.

Experimental Section
Primary Cells: The muscle tissues of two common minke whales (Moo2: immature male of 5.1m in body length; Moo3: immature female of 5.1m in body length) were obtained by sampling around Sendai Bay through the Japanese Whale Research Program under Special Permit in the western North Pacific Phase II (JAPANII) in 2015.Lethal sampling of whales was conducted under a permit issued by the Government of Japan by the provisions of Article VIII of the International Convention for the Regulation of Whaling. [35]To reduce the contamination of bacteria, the surface of tissues were first treated with 70% ethanol.Primary cell culture was performed in Dulbecco's modified Eagle medium (DMEM)/F12 medium supplemented with 10% fetal calf serum and antibiotics.The 100units mL −1 Penicillin G, 100 μg mL −1 streptomycin sulfate, 0.25 μg mL −1 Amphotericin B were used as the antibiotics.Tissue was simply shred with the ophthalmic scissors.Primary cells were preserved in a liquid nitrogen tank until use.Ethics Approval: The lethal sampling of whales was conducted in the JARPNII based on a permit issued by the Government of Japan in accordance with the provisions of Article VIII of the International Convention for the Regulation of Whaling will be stated. [36]The outline and licensing of research of this project was published in the corresponding document. [36]In this project, capture method for minke whales can be classified into two steps.Initial step was the sacrifice of animal with the penthrite grenade harpoon.Furthermore, to ensure the euthanasia of the animals, the lance was used to reduce the pain of the animals.The international agreement in IWC (International Whaling Commission) about the prohibition of cold grenade harpoon use in 1981 was exactly followed. [37]For the replacement viewpoint of animal experiments, the establishment of whale derived cells would reduce the number of sacrifices for the research work, the established immortalized cell might be significant replacement.From this view point, it has to be mentioned that this study was carried out under the ARRIVE guideline. [38]rotocol Authorization: First, Article VIII of international regulation gave the permission of whaling to Japan government (International Convention for the Regulation of Whaling (ICRW)).Furthermore, the detailed protocol for euthanasia was authorized by Scientific Committee of IWC (International Whaling Commission, Impington, United Kingdom).The detailed contents of the protocol was published as the corresponding report. [35]The inspection and authorization by the scientific commit-tee of IWC was carried out every year.Furthermore, the of the protocol was approved at a review meeting held in January 2009 and February 2016. [39]ntroduction of the Mutant CDK4, Cyclin D1, and TERT by Lentivirus-Mediated Gene Transfer: Recombinant lentiviruses expressing the R24C mutant of CDK4, cyclin D1, and TERT were produced by co-transfection of lentivirus vector plasmids CSII-CMV-CDK4R24C, CSII-CMV-cyclin D1, and CSII-CMV-TERT with the packaging plasmids, pCAG-HIV-gp and pCAG-VSVG-Rsv-Rev into 293T cells as described previously. [40]To monitor the infection efficiency of lentivirus, two lentivirus plasmids were used, CSII-CMV-EGFP and CSII-CMV-KO (Kusabira Orange).The concentrated viruses were inoculated into primary cells.

Calculation of Population Doublings (PD):
The population doubling of the cells were examined to monitor cell immortalization, as described previously. [28]Briefly, the cells were seeded at a concentration of 5.0 × 10 5 cells in a 35 mm dish in triplicate experiments.On day 1 of after the cells reached confluence, the cell numbers were counted after trypsin treatment.The PD value was measured using the formula log 2 (a/b) to determine the number of cell divisions in each passage, "a" was the final cell count at each passage, whereas "b" was the cell number seeded at the beginning of the passage.
Cell Cycle Analysis: Cell cycle analysis was performed at passages 6 or 7.The cell suspension solutions were fixed with a 70% ethanol solution, and the fixed cells were stained with the Muse Cell Cycle Kit and Analyzer.The principle of the kit was the staining with Propidium Iodide and RNase treatment.Each sample group was analyzed in six replicates, and statistical significance was evaluated using the steel method.
RNA-Seq and Downstream Analysis: Total RNA from each cell was extracted using the Nucleospin RNA extraction kit when the PD value reached 20, based on the protocol provided by the manufacturer.The quality of total RNA was evaluated by tapestation, and the cDNA libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England BioLabs).In this study, the three samples were used as biological replications, if possible, larger number of replications, such as five or seven replications is much better.However, in this study, the number of biological replications could not be increased by more than three.Three biological replications were minimum sample size to obtain the variance on statistics.Library quality and quantity were determined using an Agilent 2100 Bioanalyzer and KAPA Library Quantification Kit (KAPA Biosystems), respectively.The indexed libraries were sequenced on a NextSeq 1000 platform (Illumina) with 300 cycles in the paired-end mode.Sequence data were submitted to the DNA data bank of Japan (DDBJ) Sequence Read Archive under accession number PRJDB13430 (https://ddbj.nig.ac.jp/resource/bioproject/PRJDB13430).The total reads obtained from the machine were processed using PEAT to remove sequencing adaptors.The low quality reads were removed by the processing with FASTP.The quality of the reads was evaluated using FASTQC.
Although mapped reads were tried first using the STAR program, the reference genome sequence of the common minke whales (Balaenoptera acutorostrata, BalAcu1.0) did not have a gtf file as a reference.Thus, the reads were mapped against the Blue whole genome sequence (Balaenoptera musculus, mBalMus1.pri.v3) with STAR.The expression counts of genes were also determined using FeatureCounts.For the stable detection and accurate quantitation, genes were filtered at least 1000 counts of any sample.The specifically down or up regulated genes were detected by pairwise comparison using EdgeR with P value of zero, based on the comparison between parental cell-SV40 cell, and parental cell-K4DT cell.PCA and heatmap analyses were performed using TCC-GUI.Furthermore, the specifically down or up regulated genes were processed for pathway analysis using DAVID (https://david.ncifcrf.gov/tools.jsp).

Identification of Cell Origin of Parental Cell with RNA-Seq:
To address the cell origin of parental primary cells, the RNA-Seq comparison was carried out with SRA database.The RNA-Seq data of brain(SRR918699), heart(SRR918701), lung(SRR922125), kidney(SRR919295), liver(SRR919296), muscle(SRR922171, SRR922446) of Balaenoptera acutorostrata, and fibroblasts of Balaenoptera musculus(SRR11045090) were downloaded from the database.For the comparison of the whole RNA-transcriptome, principle components analysis (PCA) was carried out with TCC-GUI.

Figure 1 .
Figure 1.Detection of gene introduction efficiency with Kusabira-Orange expressing lentivirus and cell morphology after the infection of recombinant viruses.A) Detection of Kusabira-orange expressing in common minke whale derived Moo2 primary cells (Upper two line is low magnification, the lower line is lower magnification) Control is no infection.B) cell morphology of K4D and K4DT cells from Moo2 and Moo3 common minke whale-derived cells, and its parental cells.

Figure 2 .
Figure 2. Detection of EGFP fluorescence and KO fluorescence after the mixture of two different fluorescence expressing lentiviruses for the evaluation of multiple infection.Detection of EGFP and KO fluorescence under the microscope in the parental Moo2 cells.

Figure 3 .
Figure 3. Detection of genomic insertion and protein expression from cassettes of CDK4, cyclin D1, TERT.A) Detection of genomic insertion of expression cassettes of CDK4, cyclin D1, TERT, and internal control (Tuberous Sclerosis Type II gene).B) Western blots analysis of anti-CDK4, anticyclin D1.Anti-alpha-tubulin was used as the internal control.

Figure 4 .
Figure 4. Cell cycle analysis of flow cytometrical analysis.A) Representative results of flow cytometrical analysis of Moo3 derived parental, Moo3 derived K4D, and Moo3 derived K4DT cells.The results from parental and K4DT cell of Moo2 were also listed.B) Statistical analysis from six replications of cell cycle analysis.Statistical significance was evaluated with the Steel method.Two stars means statistical significance with a 1% level.

Figure 5 .
Figure 5. Sequential passages and detection of senescence-associated beta-galactosidase staining.A) Sequential passage of parental, K4D, and K4DT cells of Moo2 and Moo3.B) senescence results associated beta-galactosidase staining of parental cells, K4D, and K4DT cells.The staining results were obtained at passage 2(Moo2 and Moo3).C) The staining results obtained in passage 7. The staining obtained from K4D and K4DT cells were shown.The arrowheads showed positive staining.D) Semi quantitative analysis of positive incidence of SA--gal staining.The positive ratio was obtained from randomly selected three different microscope observation at X10 objective lens.The bar is standard error.Since we obtained from three replication, statistical analysis has not applied to the data.
Figure 5. Sequential passages and detection of senescence-associated beta-galactosidase staining.A) Sequential passage of parental, K4D, and K4DT cells of Moo2 and Moo3.B) senescence results associated beta-galactosidase staining of parental cells, K4D, and K4DT cells.The staining results were obtained at passage 2(Moo2 and Moo3).C) The staining results obtained in passage 7. The staining obtained from K4D and K4DT cells were shown.The arrowheads showed positive staining.D) Semi quantitative analysis of positive incidence of SA--gal staining.The positive ratio was obtained from randomly selected three different microscope observation at X10 objective lens.The bar is standard error.Since we obtained from three replication, statistical analysis has not applied to the data.

Figure 6 .
Figure 6.RNA-seq analysis of parental, SV40, K4DT cells.A) Workflow of RNA-Seq analysis.B) The correlation matrix of RNA-Seq data.C) Number of reads and mapping ratio.D) Results of 3D PCA of RNA-Seq data to address the cell origin of parental cell.E) Results of 3D PCA of RNA-Seq data of parental cell, K4DT cell, SV40 cell.F) Pairwise comparison of expression profiling to identify K4DT and SV40 specifically up or down regulated genes.

Figure 7 .
Figure 7. Heatmap analysis of K4DT specifically down-regulated genes, part1.The red color is highly expressed genes, white is middle, and blue is low expressed genes.

Figure 8 .
Figure 8. Heatmap analysis of K4DT specifically down-regulated genes, part2.The red color is highly expressed genes, white is middle, and blue is low expressed genes.

Figure 9 .
Figure 9. Heatmap analysis of K4DT specifically up-regulated genes, part1.The red color is highly expressed genes, white is middle, and blue is low expressed genes.

Figure 10 .
Figure10.Heatmap analysis of K4DT specifically up-regulated genes, part2.The red color is highly expressed genes, white is middle, and blue is low expressed genes.

Figure 11 .
Figure11.Heatmap analysis of SV40 specifically down-regulated genes, part1.The red color is highly expressed genes, white is middle, and blue is low expressed genes.

Figure 12 .
Figure12.Heatmap analysis of SV40 specifically down-regulated genes, part2.The red color is highly expressed genes, white is middle, and blue is low expressed genes.

Figure 16 .
Figure 16.Analysis of differentially expressed genes in K4DT cell on Choline metabolism in cancer pathway.Down arrowhead is down-regulated gene.Upper arrowhead is up-regulated gene.

Figure 17 .
Figure 17.Analysis of differentially expressed genes in K4DT cell on SNARE interactions in vesicular transport pathway.Down arrowhead is downregulated gene.Upper arrowhead is up-regulated gene.

Figure 18 .
Figure 18.Analysis of differentially expressed genes in K4DT cell on Longevity regulating pathway -multiple species.Down arrowhead is down-regulated gene.Upper arrowhead is up-regulated gene.

Figure 19 .
Figure 19.Analysis of differentially expressed genes in SV40 cell on Endocytosis pathway.Down arrowhead is down-regulated gene.Upper arrowhead is up-regulated gene.

Figure 20 .
Figure 20.Analysis of differentially expressed genes in SV40 cell on Hippo signaling pathway.Down arrowhead is down-regulated gene.Upper arrowhead is up-regulated gene.

Figure 21 .
Figure 21.Analysis of differentially expressed genes in SV40 cell on Human immunodeficiency virus 1 infection pathway.Down arrowhead is downregulated gene.Upper arrowhead is up-regulated gene.