Combinatorial expression of cell cycle regulators is more suitable for immortalization than oncogenic methods in dermal papilla cells

•Expression profiles of immortalized dermal papilla cells by the SV40, E6/E7, K4DT method were analyzed•The distance from wild type to was close, E6E7 middle, SV40 most distant•K4DT showed conservative profile when it compared with and The cell is an essential research tool that uses robust growth properties for functional investigation gene products. Immortalized mammalian have mainly been established using three methods: expression simian vacuolating virus 40 T antigen (the method); human papilloma virus-derived oncoprotein E6/E7 or combinatorial R24C mutant cyclin-dependent kinase 4, cyclin D1, telomerase reverse transcriptase method). However, unclear as which optimal in vitro model. Here, we biological characteristics genome-wide generated method. To our knowledge, this first study comprehensively compare determine immortalization maintaining original nature wild-type cells. These data would be valuable scientists aiming establish new lines. 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Increased arrest mammary epithelial extended capacity inactivation.Oncogene. 1998; 17: 199-205Crossref (225) accumulated cytoplasmic binds endogenous decreased CDK4-cyclin complex. reduced complex hypo-phosphorylation protein, decrease level free binding. constitutively elevated CDK4-cyclinD1 point mutation R25C enhances fibroblasts, corneal (Fukuda 2019aFukuda Gouko R. Eitsuka Suzuki Takahashi Nakagawa Sugano Tomita Human-derived expressing resource ocular toxicity testing.Front. Genet. 10: 587Crossref (8) (DPCs) 2020Fukuda Takase Orimoto A. derived constant testosterone receptor.Front. Dev. 2020; 8Crossref (3) An important characteristic intact gene, enables expansion instability. based genes used. shown works wide variety animal bovine, swine (Donai 2014Donai Guo Kuroda Sone Isogai Fukuda Bovine porcine can karyotype dependent D, telomerase.J. Biotechnol. 2014; 176: 50-57Crossref prairie vole, monkey (Kuroda 2015Kuroda Donai Establishment lines genus macaca controlled regulators.J. 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As Figure S1, although efficiency introduction only few percent, selecting successfully antibiotics (G418 hygromycin E6/E7), expressed E6/E7. previously reported 4 (CDK4), Hereafter, refer TERT K4DT. 1A, show morphology methods. Although uniform size, there variation size checked successful insertion polymerase chain reaction (PCR) analysis. Figures S2 S34, did not any insertion, while expected combinations cassettes. detected E7, tubulin western blotting. 1B S35, intense signals proteins, corresponding proteins. next analyzed cycles type, wild-type, K4DT, abnormalities, exhibited broader G2/M peak suggesting abnormalities (Figure 2).Figure 2Cell analysis DPCsShow full captionA representative histogram shown.View Large Image ViewerDownload Hi-res image Download (PPT) shown. pattern carried out RNA-Seq Illumina Hiseq platform (100 bp paired-end). workflow sequencing 3A. removal adapter sequences, mapping sequences. amount reads at least 22 M reads, enough transcriptome 3B) 2019bFukuda Doi Kobayashi Hirano Nishimori Yasue Data descriptor: Global pluripotent stem six four reprogramming factors.Sci. Data. 6: 190034Crossref (5) evaluate reproducibility data, triplicate replications. quality determined TASTQC program (Figures S3–S6). Almost all read existed within area, reliable. mapped up-to-date reference (GRCh38) STAR program. ratio more than 95%, appropriate detecting 3B). (GRh38) 96.2% (wild 1), 96.7% 2), 96.3% 3), 96.5% (K4DT-1), 96.1% (K4DT-2), 95.8% (K4DT-3), 95.1% (K4DT AR-1), AR-2), 96.4% AR-3), (SV40-1), (SV40-2), 95.9% (SV40-3), 96.8% (E6E7-1), (E6E7-2), 96.6% (E6E7-3). We, furthermore, total library cDNA (Homo_sapiens.GRCh38.cdna.all.fa) bowtie2 overall alignment rate below. 89.79% 1, 26838188 reads), 90.28% 2, 25388984 90.11% 3, 27158793 90.74% (K4DT-1, 23884949 (K4DT-2, 25895869 90.14% (K4DT-3, 26615408 88.95% AR-1, 29396085 90.30% AR-2, 26536855 reads) 88.04% AR-3, 24322764 90.40% (SV40-1, 23171547 90.49% (SV40-2, 31207937 90.71% (SV40-3, 39095454 92.24% (E6E7-1, 23119054 92.57% (E6E7-2, 26509457 91.38% (E6E7-3, 28433365 reads). complete list counts listed Figshare (https://figshare.com/s/124ed9cd873719d6cb4d). inputted TCC-GUI downstream Due TCC-GUI, used top 25,000 draw dendrogram principal component (PCA) plot explain variance level. First, 3C S7, correlation among replicates reproducible detection S7). processed three-dimensional PCA. From smallest, moderate, largest 3D). detailed locations PCA movie file 360). Three-dimensional cumulative proportion 65.5% (PC1, 33.26%, PC2, 18.964%, PC3, 13.315%), 3D whole-gene concluded closest https://www.cell.com/cms/asset/923fcc2d-6cec-4085-8a11-05e7af15f045/mmc2.mp4Loading ... .mp4 (4.02 MB) Help files Figure360.An author presentation 3Movie profiling Using (Su 2019Su W. Sun Kadota TCC-GUI: Shiny-based differential count data.BMC Notes. 12: 133Crossref (27) 33,122 (https://doi.org/10.6084/m9.figshare.12197691.v2). During differentially (DE) genes, edgeR extracted 15,103 candidates (p value less 0.01). Before applying DAVID analysis, selection DE threshold (no filtering, 300, 500, 700, 900, 1000, 1500, 1700, 2000, 2500, 3000, 10,000 counts) under p 0.01. number selected above mentioned summarized Table 1. changed no filtering 576 sample. (https://doi.org/10.6084/m9.figshare.13174148.v1), 300 (https://doi.org/10.6084/m9.figshare.13174142.v1), 500 (https://doi.org/10.6084/m9.figshare.13174151.v1), 700 (https://doi.org/10.6084/m9.figshare.13174154.v1), 900 (https://doi.org/10.6084/m9.figshare.13174157.v1), 1000 (https://doi.org/10.6084/m9.figshare.13174166.v2), 1500 (https://doi.org/10.6084/m9.figshare.13174172.v1), 1700 (https://doi.org/10.6084/m9.figshare.13174175.v1), 2000 (https://doi.org/10.6084/m9.figshare.13174178.v1), 2500 (https://doi.org/10.6084/m9.figshare.13174202.v1), 3000 (https://doi.org/10.6084/m9.figshare.13174205.v1), (https://doi.org/10.6084/m9.figshare.13174229.v1) given.Table 1Number thresholds cellsFilteringNumber genesFile nameNo < 0.01 EdgeR15,103f_list1_1.csv300 sample, EdgeR9836f_list1_2.csv500 P EdgeR8651f_list1_3.csv700 EdgeR8036f_list1_4.csv900 EdgeR6750f_list1_5.csv1000 EdgeR6350f_list1_6.csv1500 EdgeR4829f_list1_7.csv1700 EdgeR4355f_list1_8.csv2000 EdgeR3786f_list1_9.csv2500 EdgeR3060f_list1_10.csv3000 EdgeR2524f_list1_11.csv10,000 EdgeR547f_list1_12.csv Open table tab counts), pathway each threshold. 5 significant 2. times highest (10 times), ribosome (9 (5 times). viewpoint association cancer.Table 2List five analyses cells.FilteringNumber genesDAVID ranking1st2nd3rd4th5thNo EdgeR15,103Pathway cancerSmall lung cancerNeurotrophin signaling pathwayChronic myeloid leukemiaEndocytosis300 EdgeR9836Cell cycleColorecal cancerPancreatic cancerChronic leukemiaPyrimidine metabolism500 EdgeR8651Cell cyclesmall cancerSpliceosomeRibosomePancretic cancer700 EdgeR8036RibosomeCell cycleUbiquitin mediated proteolysisChronic leukemiaSpliceosome900 EdgeR6750Cell cycleRibosomeSpliceosomeUbiquitin myeloidleukemia1000 EdgeR6350RibosomeCell cycleSpliceosomeProteasomeFocal adhesion1500 EdgeR4829RibosomeCell cycleFocal adhesionProteasomePathway cancer1700 EdgeR4355RibosomeFocal adhesionCell cycleProteasomePathway cancer2000 EdgeR3786RibosomeFocal adhesionProteasomeCell cyclePathway cancer2500 EdgeR3060RibosomeFocal adhesionProteasomeRegulation actine cytoskeletonPathway cancer3000 EdgeR2524RibosomeFocal adhesionECM-receptorInteractionRegulation actin cytoskeletonCell cycle10,000 EdgeR547RibosomeFocal adhesionECM-receptor interactionPathogenic Escherichia coli infectionArrhythmogenic right ventricular cardiomyopathy (ARVC)ECM, Extracellular Matrix ECM, corresponded (112 genes). levels 112 after normalization, presented heatmap 5A. 4A, normalized reveals difference between small, differences raw cycle-related KEGG box plots S8–S14. change 2X greater, 0.5X less, map 5). 6, affected restricted D-CDK4 complex-related networks, signals, c-Myc, E-CDK2, B-CDK1, APC/C-Cdc complex.Figure 5Summary upregulated downregulated genesShow captionArrowheads indicate 200 X2 increase 1/2 mapped.View (PPT)Figure 6Summary cancer-related Arrowheads mapped. cancer. contains 282 genes. 4B. 4B, relatively close S15–S32. 6). Mapping revealed c-Jun, c-Fos, locate MAP signaling, has unregulated FGF FGF-receptor Lastly, chromosomes had normal 46 50 mitotic broad distribution numbers, 71 80, chromosomal abnormalities. 10% further G banding (Table S2). one translocation 14 15, 95%. 0%, 20% chromosomes. results, caused fewest DPCs. contrast, minor moderate 7B). oncoprotein, enabled comprehensively. suitable HEK293T assays mimic vivo. wish use condition. Our manuscript demonstrated immortalizing retaining nature, attractive trying select modeling anti-proliferative p16/tumor gate replicative DAVID, nucleotide excision repair plays maintenance genome. view point, S33). good agreement turnover, upregulation including PCNA epsilon detected. TFIIH complex-, XPE XPC complex-associated molecules consistent suggest Potential abnormality need about accuracy androgen receptor (AR) transfer. exact mechanism unclear, AR lower several passages (Kwack 2008Kwack M.H. Sung Y.K. Chung E.J. Im S.U. Ahn J.S. Kim M.K. J.C. Dihydrotestosterone-inducible dickkopf 1 balding apoptosis follicular keratinocytes.J. Invest. Dermatol. 2008; 128: 262-269Abstract (136) previous reports suppression, downregulation now analyze AR. comparison AR-expressing K4DTs parent pathway. better signal network beneficial anti-testosterone drugs treat prostate androgenic alopecia, activation status sampling passage 3 while, finished 7 8. sample collection 6 (see section finish up lines, extremely evaluation. triplicates might concise conclusion. precise evaluation larger duplication necessary address node Further information requests resources vectors should directed fulfilled Lead Contact, Tomokazu ( [email protected] ). sets next-generation sequencer article uploaded Bank Japan (DDBJ, assigned BioProject accession SSUB015042. output lists Figshare. given. Software versions FastQC, version 0.11.8, check FASTQ file. https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ PRINSEQ, 0.20.4, low reads. http://prinseq.sourceforge.net/ PEAT, 1.2, https://github.com/jhhung/PEAT. STAR, 2.6.1, mapping. https://github.com/alexdobin/STAR featureCount, SUBREAD, release 1.6.5, counting. http://subread.sourceforge.net. Bowtie2, 2.3.4.3, (Homo_sapiens.GRCh38.cdna.all.fa). http://bowtie-bio.sourceforge.net/bowtie2/index.shtml. R package, 4.0.3, https://www.r-project.org. https://github.com/swsoyee/TCC-GUI. shared material transfer agreements technology office authors. All found accompanying Transparent supplemental thank Dr. Hiroyuki Miyoshi (RIKEN, BioResource Center) providing packaging plasmids recombinant virus. Taku Ozaki (Graduate School Science Technology, Iwate University) mentoring students. Koji (Tokyo kind help overcome problem Mr. Ryoji Mss. Hisako encouragement supports throughout study. T.F., E.S., H.T., S.K., Y.T., T.I., contributed experimental design. K.F., K.T., A.O., performed experiments. T.K. construction supported T.F. wrote manuscript. authors declare conflict interests. .pdf (26.68 pdf Document S1.Transparent S1–S33