Characterisation of the novel spontaneously immortalized and invasively growing human skin keratinocyte line HaSKpw.
Carcinogenesis
Cell Line, Tumor
/ metabolism
Cell Movement
Clone Cells
Gene Expression Regulation, Neoplastic
HaCaT Cells
Humans
Keratinocytes
/ physiology
Keratins, Hair-Specific
/ genetics
Keratins, Type I
/ genetics
MicroRNAs
/ genetics
Neoplasm Invasiveness
Protein-Lysine 6-Oxidase
/ genetics
S100 Proteins
/ genetics
Skin
/ pathology
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
16 09 2020
16 09 2020
Historique:
received:
16
01
2020
accepted:
10
08
2020
entrez:
17
9
2020
pubmed:
18
9
2020
medline:
8
1
2021
Statut:
epublish
Résumé
We here present the spontaneously immortalised cell line, HaSKpw, as a novel model for the multistep process of skin carcinogenesis. HaSKpw cells were established from the epidermis of normal human adult skin that, without crisis, are now growing unrestricted and feeder-independent. At passage 22, clonal populations were established and clone7 (HaSKpwC7) was further compared to the also spontaneously immortalized HaCaT cells. As important differences, the HaSKpw cells express wild-type p53, remain pseudodiploid, and show a unique chromosomal profile with numerous complex aberrations involving chromosome 20. In addition, HaSKpw cells overexpress a pattern of genes and miRNAs such as KRT34, LOX, S100A9, miR21, and miR155; all pointing to a tumorigenic status. In concordance, HaSKpw cells exhibit reduced desmosomal contacts that provide them with increased motility and a highly migratory/invasive phenotype as demonstrated in scratch- and Boyden chamber assays. In 3D organotypic cultures, both HaCaT and HaSKpw cells form disorganized epithelia but only the HaSKpw cells show tumorcell-like invasive growth. Together, HaSKpwC7 and HaCaT cells represent two spontaneous (non-genetically engineered) "premalignant" keratinocyte lines from adult human skin that display different stages of the multistep process of skin carcinogenesis and thus represent unique models for analysing skin cancer development and progression.
Identifiants
pubmed: 32938951
doi: 10.1038/s41598-020-71315-0
pii: 10.1038/s41598-020-71315-0
pmc: PMC7494900
doi:
Substances chimiques
KRT34 protein, human
0
Keratins, Hair-Specific
0
Keratins, Type I
0
MIRN155 microRNA, human
0
MIRN21 microRNA, human
0
MicroRNAs
0
S100 Proteins
0
S100A1 protein
0
LOX protein, human
EC 1.4.3.13
Protein-Lysine 6-Oxidase
EC 1.4.3.13
Types de publication
Comparative Study
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
15196Références
Maqsood, M. I., Matin, M. M., Bahrami, A. R. & Ghasroldasht, M. M. Immortality of cell lines: challenges and advantages of establishment. Cell Biol. Int. 37, 1038–1045. https://doi.org/10.1002/cbin.10137 (2013).
doi: 10.1002/cbin.10137
pubmed: 23723166
Steinberg, M. L. & Defendi, V. Altered pattern of growth and differentiation in human keratinocytes infected by simian virus 40. Proc. Natl. Acad. Sci. U. S. A. 76, 801–805. https://doi.org/10.1073/pnas.76.2.801 (1979).
doi: 10.1073/pnas.76.2.801
pubmed: 218222
pmcid: 383055
Taylor-Papadimitriou, J., Purkis, P., Lane, E. B., McKay, I. A. & Chang, S. E. Effects of SV40 transformation on the cytoskeleton and behavioural properties of human keratinocytes. Cell Differ. 11, 169–180 (1982).
doi: 10.1016/0045-6039(82)90008-2
Banks-Schlegel, S. P. & Howley, P. M. Differentiation of human epidermal cells transformed by SV40. J. Cell Biol. 96, 330–337. https://doi.org/10.1083/jcb.96.2.330 (1983).
doi: 10.1083/jcb.96.2.330
pubmed: 6187748
Brown, K. W. & Parkinson, E. K. Extracellular matrix components produced by SV40-transformed human epidermal keratinocytes. Int. J. Cancer 33, 257–263 (1984).
doi: 10.1002/ijc.2910330215
Agarwal, C., Rorke, E. A., Irwin, J. C. & Eckert, R. L. Immortalization by human papillomavirus type 16 alters retinoid regulation of human ectocervical epithelial cell differentiation. Cancer Res. 51, 3982–3989 (1991).
pubmed: 1713124
Hurlin, P. J. et al. Progression of human papillomavirus type 18-immortalized human keratinocytes to a malignant phenotype. Proc. Natl. Acad. Sci. U. S. A. 88, 570–574. https://doi.org/10.1073/pnas.88.2.570 (1991).
doi: 10.1073/pnas.88.2.570
pubmed: 1846447
pmcid: 50853
Lee, K. M., Choi, K. H. & Ouellette, M. M. Use of exogenous hTERT to immortalize primary human cells. Cytotechnology 45, 33–38. https://doi.org/10.1007/10.1007/s10616-004-5123-3 (2004).
doi: 10.1007/10.1007/s10616-004-5123-3
pubmed: 19003241
pmcid: 3449956
Allen-Hoffmann, B. L. et al. Normal growth and differentiation in a spontaneously immortalized near-diploid human keratinocyte cell line, NIKS. J. Investig. Dermatol. 114, 444–455. https://doi.org/10.1046/j.1523-1747.2000.00869.x (2000).
doi: 10.1046/j.1523-1747.2000.00869.x
pubmed: 10692102
Kress, T. R., Sabo, A. & Amati, B. MYC: Connecting selective transcriptional control to global RNA production. Nat. Rev. Cancer 15, 593–607. https://doi.org/10.1038/nrc3984 (2015).
doi: 10.1038/nrc3984
pubmed: 26383138
Boukamp, P. et al. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J. Cell Biol. 106, 761–771. https://doi.org/10.1083/jcb.106.3.761 (1988).
doi: 10.1083/jcb.106.3.761
pubmed: 2450098
Nakazawa, H. et al. UV and skin cancer: Specific p53 gene mutation in normal skin as a biologically relevant exposure measurement. Proc. Natl. Acad. Sci. U. S. A. 91, 360–364. https://doi.org/10.1073/pnas.91.1.360 (1994).
doi: 10.1073/pnas.91.1.360
pubmed: 8278394
pmcid: 42947
Miyata, Y. et al. The effect of the long-term cultivation on telomere length and morphology of cultured epidermis. J. Dermatol. Sci. 34, 221–230. https://doi.org/10.1016/j.jdermsci.2004.02.004 (2004).
doi: 10.1016/j.jdermsci.2004.02.004
pubmed: 15113592
Harle-Bachor, C. & Boukamp, P. Telomerase activity in the regenerative basal layer of the epidermis inhuman skin and in immortal and carcinoma-derived skin keratinocytes. Proc. Natl. Acad. Sci. U. S. A. 93, 6476–6481. https://doi.org/10.1073/pnas.93.13.6476 (1996).
doi: 10.1073/pnas.93.13.6476
pubmed: 8692840
pmcid: 39048
Krunic, D. et al. Tissue context-activated telomerase in human epidermis correlates with little age-dependent telomere loss. Biochim. Biophys. Acta 1792, 297–308. https://doi.org/10.1016/j.bbadis.2009.02.005 (2009).
doi: 10.1016/j.bbadis.2009.02.005
pubmed: 19419690
Cerezo, A., Kalthoff, H., Schuermann, M., Schafer, B. & Boukamp, P. Dual regulation of telomerase activity through c-Myc-dependent inhibition and alternative splicing of hTERT. J. Cell Sci. 115, 1305–1312 (2002).
pubmed: 11884529
Khosravi-Maharlooei, M. et al. Expression pattern of alternative splicing variants of human telomerase reverse transcriptase (hTERT) in cancer cell lines was not associated with the origin of the cells. Int. J. Mol. Cell Med. 4, 109–119 (2015).
pubmed: 26261800
pmcid: 4499573
Boukamp, P. et al. Sustained nontumorigenic phenotype correlates with a largely stable chromosome content during long-term culture of the human keratinocyte line HaCaT. Genes Chromosom. Cancer 19, 201–214 (1997).
doi: 10.1002/(SICI)1098-2264(199708)19:4<201::AID-GCC1>3.0.CO;2-0
Boukamp, P., Stanbridge, E. J., Foo, D. Y., Cerutti, P. A. & Fusenig, N. E. c-Ha-ras oncogene expression in immortalized human keratinocytes (HaCaT) alters growth potential in vivo but lacks correlation with malignancy. Cancer Res. 50, 2840–2847 (1990).
pubmed: 2183932
Benjamin, C. L. & Ananthaswamy, H. N. p53 and the pathogenesis of skin cancer. Toxicol. Appl. Pharmacol. 224, 241–248. https://doi.org/10.1016/j.taap.2006.12.006 (2007).
doi: 10.1016/j.taap.2006.12.006
pubmed: 17270229
Lehman, T. A. et al. p53 mutations in human immortalized epithelial cell lines. Carcinogenesis 14, 833–839. https://doi.org/10.1093/carcin/14.5.833 (1993).
doi: 10.1093/carcin/14.5.833
pubmed: 8504475
Whibley, C., Pharoah, P. D. & Hollstein, M. p53 polymorphisms: Cancer implications. Nat. Rev. Cancer 9, 95–107. https://doi.org/10.1038/nrc2584 (2009).
doi: 10.1038/nrc2584
pubmed: 19165225
Najor, N. A. Desmosomes in human disease. Annu. Rev. Pathol. 13, 51–70. https://doi.org/10.1146/annurev-pathol-020117-044030 (2018).
doi: 10.1146/annurev-pathol-020117-044030
pubmed: 29414250
Moch, M., Schwarz, N., Windoffer, R. & Leube, R. E. The keratin-desmosome scaffold: Pivotal role of desmosomes for keratin network morphogenesis. Cell Mol. Life Sci. 77, 543–558. https://doi.org/10.1007/s00018-019-03198-y (2020).
doi: 10.1007/s00018-019-03198-y
pubmed: 31243490
Bazzi, H. et al. Desmoglein 4 is expressed in highly differentiated keratinocytes and trichocytes in human epidermis and hair follicle. Differentiation 74, 129–140. https://doi.org/10.1111/j.1432-0436.2006.00061.x (2006).
doi: 10.1111/j.1432-0436.2006.00061.x
pubmed: 16533311
Jurcic, V., Kukovic, J. & Zidar, N. Expression of desmosomal proteins in acantholytic squamous cell carcinoma of the skin. Histol. Histopathol. 30, 945–953. https://doi.org/10.14670/HH-11-599 (2015).
doi: 10.14670/HH-11-599
pubmed: 25723181
Krunic, A. L., Garrod, D. R., Smith, N. P., Orchard, G. S. & Cvijetic, O. B. Differential expression of desmosomal glycoproteins in keratoacanthoma and squamous cell carcinoma of the skin: An immunohistochemical aid to diagnosis. Acta Derm. Venereol. 76, 394–398. https://doi.org/10.2340/0001555576394398 (1996).
doi: 10.2340/0001555576394398
pubmed: 8891017
Wang, L. H. Molecular signaling regulating anchorage-independent growth of cancer cells. Mt Sinai J. Med. 71, 361–367 (2004).
pubmed: 15592654
Boukamp, P., Rupniak, H. T. & Fusenig, N. E. Environmental modulation of the expression of differentiation and malignancy in six human squamous cell carcinoma cell lines. Cancer Res. 45, 5582–5592 (1985).
pubmed: 4053033
Korver, J. E., van Duijnhoven, M. W., Pasch, M. C., van Erp, P. E. & van de Kerkhof, P. C. Assessment of epidermal subpopulations and proliferation in healthy skin, symptomless and lesional skin of spreading psoriasis. Br. J. Dermatol. 155, 688–694. https://doi.org/10.1111/j.1365-2133.2006.07403.x (2006).
doi: 10.1111/j.1365-2133.2006.07403.x
pubmed: 16965416
Thewes, M., Stadler, R., Korge, B. & Mischke, D. Normal psoriatic epidermis expression of hyperproliferation-associated keratins. Arch. Dermatol. Res. 283, 465–471 (1991).
doi: 10.1007/BF00371784
Herrmann, H., Bar, H., Kreplak, L., Strelkov, S. V. & Aebi, U. Intermediate filaments: From cell architecture to nanomechanics. Nat. Rev. Mol. Cell Biol. 8, 562–573. https://doi.org/10.1038/nrm2197 (2007).
doi: 10.1038/nrm2197
pubmed: 17551517
Omary, M. B., Ku, N. O., Strnad, P. & Hanada, S. Toward unraveling the complexity of simple epithelial keratins in human disease. J. Clin. Investig. 119, 1794–1805. https://doi.org/10.1172/JCI37762 (2009).
doi: 10.1172/JCI37762
pubmed: 19587454
Krieg, P. & Furstenberger, G. The role of lipoxygenases in epidermis. Biochim. Biophys. Acta 390–400, 2014. https://doi.org/10.1016/j.bbalip.2013.08.005 (1841).
doi: 10.1016/j.bbalip.2013.08.005
Yu, G., Wang, L. G., Han, Y. & He, Q. Y. clusterProfiler: An R package for comparing biological themes among gene clusters. OMICS 16, 284–287. https://doi.org/10.1089/omi.2011.0118 (2012).
doi: 10.1089/omi.2011.0118
pubmed: 22455463
pmcid: 3339379
Yan, L. et al. MiR-21-5p links epithelial–mesenchymal transition phenotype with stem-like cell signatures via AKT signaling in keloid keratinocytes. Sci. Rep. 6, 28281. https://doi.org/10.1038/srep28281 (2016).
doi: 10.1038/srep28281
pubmed: 27596120
pmcid: 5011940
Ma, X. et al. miR-203a controls keratinocyte proliferation and differentiation via targeting the stemness-associated factor DeltaNp63 and establishing a regulatory circuit with SNAI2. Biochem. Biophys. Res. Commun. 491, 241–249. https://doi.org/10.1016/j.bbrc.2017.07.131 (2017).
doi: 10.1016/j.bbrc.2017.07.131
pubmed: 28754589
Ye, Z. B. et al. miR-429 inhibits migration and invasion of breast cancer cells in vitro. Int. J. Oncol. 46, 531–538. https://doi.org/10.3892/ijo.2014.2759 (2015).
doi: 10.3892/ijo.2014.2759
pubmed: 25405387
Berning, M., Pratzel-Wunder, S., Bickenbach, J. R. & Boukamp, P. Three-dimensional in vitro skin and skin cancer models based on human fibroblast-derived matrix. Tissue Eng. Part C Methods 21, 958–970. https://doi.org/10.1089/ten.TEC.2014.0698 (2015).
doi: 10.1089/ten.TEC.2014.0698
pubmed: 25837604
Muffler, S. et al. A stable niche supports long-term maintenance of human epidermal stem cells in organotypic cultures. Stem Cells 26, 2506–2515. https://doi.org/10.1634/stemcells.2007-0991 (2008).
doi: 10.1634/stemcells.2007-0991
pubmed: 18653773
Pavez Lorie, E., Berning, M., Boukamp, P. in Skin tissue models Vol. 1 (ed Picarro, R. et al.) Ch. 7, 151–173 (Elsevier Inc, Amsterdam, 2018).
Carless, M. A. & Griffiths, L. R. Cytogenetics of melanoma and nonmelanoma skin cancer. Adv. Exp. Med. Biol. 810, 160–181. https://doi.org/10.1007/978-1-4939-0437-2_9 (2014).
doi: 10.1007/978-1-4939-0437-2_9
pubmed: 25207365
Wang, Q. et al. Qualitative and quantitative expression status of the human chromosome 20 genes in cancer tissues and the representative cell lines. J. Proteome Res. 12, 151–161. https://doi.org/10.1021/pr3008336 (2013).
doi: 10.1021/pr3008336
pubmed: 23252959
Jin, Y. et al. Cytogenetic and molecular genetic characterization of immortalized human ovarian surface epithelial cell lines: Consistent loss of chromosome 13 and amplification of chromosome 20. Gynecol. Oncol. 92, 183–191. https://doi.org/10.1016/j.ygyno.2003.09.007 (2004).
doi: 10.1016/j.ygyno.2003.09.007
pubmed: 14751156
Tabach, Y. et al. Amplification of the 20q chromosomal arm occurs early in tumorigenic transformation and may initiate cancer. PLoS ONE 6, e14632. https://doi.org/10.1371/journal.pone.0014632 (2011).
doi: 10.1371/journal.pone.0014632
pubmed: 21297939
pmcid: 3031497
Maffei, M. et al. Chromosome 20 aberrations at the diploid-aneuploid transition in sporadic colorectal cancer. Cytogenet. Genome Res. 144, 9–14. https://doi.org/10.1159/000367909 (2014).
doi: 10.1159/000367909
pubmed: 25323042
Chang, T. H. et al. The effects of actin cytoskeleton perturbation on keratin intermediate filament formation in mesenchymal stem/stromal cells. Biomaterials 35, 3934–3944. https://doi.org/10.1016/j.biomaterials.2014.01.028 (2014).
doi: 10.1016/j.biomaterials.2014.01.028
pubmed: 24513317
Seltmann, K., Fritsch, A. W., Kas, J. A. & Magin, T. M. Keratins significantly contribute to cell stiffness and impact invasive behavior. Proc. Natl. Acad. Sci. U. S. A. 110, 18507–18512. https://doi.org/10.1073/pnas.1310493110 (2013).
doi: 10.1073/pnas.1310493110
pubmed: 24167274
pmcid: 3832002
Ridky, T. W., Chow, J. M., Wong, D. J. & Khavari, P. A. Invasive three-dimensional organotypic neoplasia from multiple normal human epithelia. Nat. Med. 16, 1450–1455. https://doi.org/10.1038/nm.2265 (2010).
doi: 10.1038/nm.2265
pubmed: 21102459
pmcid: 3586217
Padilla, R. S., Sebastian, S., Jiang, Z., Nindl, I. & Larson, R. Gene expression patterns of normal human skin, actinic keratosis, and squamous cell carcinoma: A spectrum of disease progression. Arch. Dermatol. 146, 288–293. https://doi.org/10.1001/archdermatol.2009.378 (2010).
doi: 10.1001/archdermatol.2009.378
pubmed: 20231500
Iotzova-Weiss, G. et al. S100A8/A9 stimulates keratinocyte proliferation in the development of squamous cell carcinoma of the skin via the receptor for advanced glycation-end products. PLoS ONE 10, e0120971. https://doi.org/10.1371/journal.pone.0120971 (2015).
doi: 10.1371/journal.pone.0120971
pubmed: 25811984
pmcid: 4374726
Gebhardt, C. et al. Calgranulins S100A8 and S100A9 are negatively regulated by glucocorticoids in a c-Fos-dependent manner and overexpressed throughout skin carcinogenesis. Oncogene 21, 4266–4276. https://doi.org/10.1038/sj.onc.1205521 (2002).
doi: 10.1038/sj.onc.1205521
pubmed: 12082614
Hameetman, L. et al. Molecular profiling of cutaneous squamous cell carcinomas and actinic keratoses from organ transplant recipients. BMC Cancer 13, 58. https://doi.org/10.1186/1471-2407-13-58 (2013).
doi: 10.1186/1471-2407-13-58
pubmed: 23379751
pmcid: 3570297
Darido, C. et al. Targeting of the tumor suppressor GRHL3 by a miR-21-dependent proto-oncogenic network results in PTEN loss and tumorigenesis. Cancer Cell 20, 635–648. https://doi.org/10.1016/j.ccr.2011.10.014 (2011).
doi: 10.1016/j.ccr.2011.10.014
pubmed: 22094257
Selcuklu, S. D., Donoghue, M. T. & Spillane, C. miR-21 as a key regulator of oncogenic processes. Biochem. Soc. Trans. 37, 918–925. https://doi.org/10.1042/BST0370918 (2009).
doi: 10.1042/BST0370918
pubmed: 19614619
Xu, N. et al. MicroRNA-125b down-regulates matrix metallopeptidase 13 and inhibits cutaneous squamous cell carcinoma cell proliferation, migration, and invasion. J. Biol. Chem. 287, 29899–29908. https://doi.org/10.1074/jbc.M112.391243 (2012).
doi: 10.1074/jbc.M112.391243
pubmed: 22782903
pmcid: 3436131
Boukamp, P. Non-melanoma skin cancer: What drives tumor development and progression?. Carcinogenesis 26, 1657–1667. https://doi.org/10.1093/carcin/bgi123 (2005).
doi: 10.1093/carcin/bgi123
pubmed: 15905207
Stahl, P. L. et al. Sun-induced nonsynonymous p53 mutations are extensively accumulated and tolerated in normal appearing human skin. J. Investig. Dermatol. 131, 504–508. https://doi.org/10.1038/jid.2010.302 (2011).
doi: 10.1038/jid.2010.302
pubmed: 20944651
Boehnke, K. et al. Effects of fibroblasts and microenvironment on epidermal regeneration and tissue function in long-term skin equivalents. Eur. J. Cell Biol. 86, 731–746. https://doi.org/10.1016/j.ejcb.2006.12.005 (2007).
doi: 10.1016/j.ejcb.2006.12.005
pubmed: 17292509
Germain, L. et al. Improvement of human keratinocyte isolation and culture using thermolysin. Burns 19, 99–104. https://doi.org/10.1016/0305-4179(93)90028-7 (1993).
doi: 10.1016/0305-4179(93)90028-7
pubmed: 8471157
Rheinwald, J. G. in Cell Growth and Division (ed Baserga, R.) (Oxford University Press, Oxford, 1989).
Darzynkiewicz, Z. & Huang, X. Analysis of cellular DNA content by flow cytometry. Curr Protoc Immunol. https://doi.org/10.1002/0471142735.im0507s60 (2004).
doi: 10.1002/0471142735.im0507s60
pubmed: 18432930
Tinevez, J. Y. et al. TrackMate: An open and extensible platform for single-particle tracking. Methods 115, 80–90. https://doi.org/10.1016/j.ymeth.2016.09.016 (2017).
doi: 10.1016/j.ymeth.2016.09.016
pubmed: 27713081
Falasca, M., Raimondi, C. & Maffucci, T. Boyden chamber. Methods Mol. Biol. 769, 87–95. https://doi.org/10.1007/978-1-61779-207-6_7 (2011).
doi: 10.1007/978-1-61779-207-6_7
pubmed: 21748671
Möller, B., Glaß, M., Misiak, D. & Posch, S. MiToBo—A toolbox for image processing and analysis. J. Open Res. Softw. https://doi.org/10.5334/jors.103 (2016).
doi: 10.5334/jors.103
Denning, M. F. et al. The expression of desmoglein isoforms in cultured human keratinocytes is regulated by calcium, serum, and protein kinase C. Exp. Cell Res. 239, 50–59. https://doi.org/10.1006/excr.1997.3890 (1998).
doi: 10.1006/excr.1997.3890
pubmed: 9511724
Keith, W. N. & Hoare, S. F. Detection of telomerase hTERT gene expression and its splice variants by RT-PCR. Methods Mol. Med. 97, 297–309. https://doi.org/10.1385/1-59259-760-2:297 (2004).
doi: 10.1385/1-59259-760-2:297
pubmed: 15064501
Kilian, A. et al. Isolation of a candidate human telomerase catalytic subunit gene, which reveals complex splicing patterns in different cell types. Hum. Mol. Genet. 6, 2011–2019. https://doi.org/10.1093/hmg/6.12.2011 (1997).
doi: 10.1093/hmg/6.12.2011
pubmed: 9328464
Cawthon, R. M. Telomere measurement by quantitative PCR. Nucleic Acids Res. 30, e47. https://doi.org/10.1093/nar/30.10.e47 (2002).
doi: 10.1093/nar/30.10.e47
pubmed: 12000852
pmcid: 115301
O’Callaghan, N. J. & Fenech, M. A quantitative PCR method for measuring absolute telomere length. Biol. Proc. Online 13, 3. https://doi.org/10.1186/1480-9222-13-3 (2011).
doi: 10.1186/1480-9222-13-3
Geigl, J. B., Uhrig, S. & Speicher, M. R. Multiplex-fluorescence in situ hybridization for chromosome karyotyping. Nat. Protoc. 1, 1172–1184. https://doi.org/10.1038/nprot.2006.160 (2006).
doi: 10.1038/nprot.2006.160
pubmed: 17406400
Dirks, W. G. & Drexler, H. G. STR DNA typing of human cell lines: Detection of intra- and interspecies cross-contamination. Methods Mol. Biol. 946, 27–38. https://doi.org/10.1007/978-1-62703-128-8_3 (2013).
doi: 10.1007/978-1-62703-128-8_3
pubmed: 23179824
Kalfalah, F. M. et al. Spatio-temporal regulation of the human licensing factor Cdc6 in replication and mitosis. Cell Cycle 14, 1704–1715. https://doi.org/10.1080/15384101.2014.1000182 (2015).
doi: 10.1080/15384101.2014.1000182
pubmed: 25875233
pmcid: 4614858
Eberwine, J., Spencer, C., Miyashiro, K., Mackler, S. & Finnell, R. Complementary DNA synthesis in situ: methods and applications. Methods Enzymol. 216, 80–100 (1992).
doi: 10.1016/0076-6879(92)16011-8
Pavez Lorie, E. & Boukamp, P. Methods in cell biology: Cell-derived matrices. Methods Cell Biol. 156, 309–332. https://doi.org/10.1016/bs.mcb.2019.11.012 (2020).
doi: 10.1016/bs.mcb.2019.11.012
pubmed: 32222225