miR-873-3p targets HDAC4 to stimulate matrix metalloproteinase-13 expression upon parathyroid hormone exposure in rat osteoblasts.
Animals
Bone Remodeling
/ genetics
Core Binding Factor Alpha 1 Subunit
/ genetics
Gene Expression Regulation, Developmental
/ genetics
Histone Deacetylases
/ genetics
Humans
Matrix Metalloproteinase 13
/ genetics
Mice
MicroRNAs
/ genetics
Osteoblasts
/ metabolism
Osteogenesis
/ genetics
Parathyroid Hormone
/ genetics
Rats
Transcriptional Activation
/ genetics
HDAC4
MMP-13
Runx2
miR-873-3p
parathyroid hormone
Journal
Journal of cellular physiology
ISSN: 1097-4652
Titre abrégé: J Cell Physiol
Pays: United States
ID NLM: 0050222
Informations de publication
Date de publication:
11 2020
11 2020
Historique:
received:
09
07
2019
accepted:
03
01
2020
pubmed:
22
1
2020
medline:
13
3
2021
entrez:
22
1
2020
Statut:
ppublish
Résumé
Matrix metalloproteinase-13 (MMP-13) plays a predominant role in endochondral bone formation and bone remodeling. Parathyroid hormone (PTH) stimulates the expression of MMP-13 via Runx2, a bone transcription factor in rat osteoblastic cells (UMR106-01), and histone deacetylase 4 (HDAC4) acts as a corepressor of Runx2. Moreover, microRNAs (miRNAs) play an important role in regulating genes posttranscriptionally. Here, we hypothesized that PTH upregulates the miRNAs targeting HDAC4, which could lead to increased Runx2 activity and MMP-13 expression in rat osteoblastic cells. We identified several miRNAs that putatively target rat HDAC4 using bioinformatics tools. miR-873-3p was significantly upregulated by PTH in rat osteoblasts. miR-873-3p overexpression downregulated HDAC4 protein expression, increased Runx2 binding at the MMP-13 promoter, and increased MMP-13 messenger RNA expression in UMR106-01 cells. A luciferase reporter assay identified the direct targeting of miR-873-3p at the 3'-untranslated region of HDAC4. Thus, miR-873-3p targeted HDAC4 and relieved the corepressor effect of HDAC4 on Runx2 for MMP-13 expression in rat osteoblasts. This study advances our knowledge of posttranscriptional gene regulation occurring in bone and bone-related diseases and clarifies the role of miRNAs as diagnostic biomarkers.
Substances chimiques
Core Binding Factor Alpha 1 Subunit
0
MIRN873 microRNA, human
0
MicroRNAs
0
Parathyroid Hormone
0
Matrix Metalloproteinase 13
EC 3.4.24.-
HDAC4 protein, rat
EC 3.5.1.98
Histone Deacetylases
EC 3.5.1.98
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
7996-8009Informations de copyright
© 2020 Wiley Periodicals, Inc.
Références
Arumugam, B., Vairamani, M., Partridge, N. C., & Selvamurugan, N. (2018). Characterization of Runx2 phosphorylation sites required for TGF-β1-mediated stimulation of matrix metalloproteinase-13 expression in osteoblastic cells. Journal of Cellular Physiology, 233(2), 1082-1094. https://doi.org/10.1002/jcp.25964
Banerjee, C., McCabe, L. R., Choi, J. Y., Hiebert, S. W., Stein, J. L., Stein, G. S., & Lian, J. B. (1997). Runt homology domain proteins in osteoblast differentiation: AML3/CBFA1 is a major component of a bone-specific complex. Journal of Cellular Biochemistry, 66(1), 1-8. https://doi.org/10.1002/(SICI)1097-4644(19970701)66:1<1::AID-JCB1>3.0.CO;2-V
Boumah, C. E., Lee, M., Selvamurugan, N., Shimizu, E., & Partridge, N. C. (2009). Runx2 recruits p300 to mediate parathyroid hormone's effects on histone acetylation and transcriptional activation of the matrix metalloproteinase-13 gene. Molecular Endocrinology, 23, 1255-1263. https://doi.org/10.1210/me.2008-0217
Carey, M. F., Peterson, C. L., & Smale, S. T. (2009). Chromatin immunoprecipitation (chip). Cold Spring Harbor Protocols, 9, pdb-rot5279. https://doi.org/10.1101/pdb.prot5279
Chou, C. H., Shrestha, S., Yang, C. D., Chang, N. W., Lin, Y. L., Liao, K. W., … Huang, H. D (2017). miRTarBase update 2018: A resource for experimentally validated microRNA-target interactions. Nucleic Acids Research, 46, D296-D302. https://doi.org/10.1093/nar/gkx1067
D'Alonzo, R. C., Selvamurugan, N., Karsenty, G., & Partridge, N. C. (2002). Physical interaction of the activator protein-1 factors c-Fos and c-Jun with Cbfa1 for collagenase-3 promoter activation. Journal of Biological Chemistry, 277(1), 816-822. https://doi.org/10.1074/jbc.M107082200
De Ruijter, A. J., Van Gennip, A. H., Caron, H. N., Kemp, S., & Van Kuilenburg, A. B. (2003). Histone deacetylases (HDACs): Characterization of the classical HDAC family. Biochemical Journal, 370(3), 737-749. https://doi.org/10.1042/BJ20021321
Dillon, J. P., Waring-Green, V. J., Taylor, A. M., Wilson, P. J., Birch, M., Gartland, A., & Gallagher, J. A. (2012). Primary human osteoblast cultures, Bone research protocols. Methods in molecular biology (816, pp. 3-18). Totowa, NJ: Humana Press. https://doi.org/10.1007/978-1-61779-415-5_1
Feng, Q., Zheng, S., & Zheng, J. (2018). The emerging role of microRNAs in bone remodeling and its therapeutic implications for osteoporosis. Bioscience Reports, 38, BSR20180453. https://doi.org/10.1042/BSR20180453
Gnecchi, M., & Melo, L. G. (2009). Bone marrow-derived mesenchymal stem cells: Isolation, expansion, characterization, viral transduction, and production of conditioned medium. Methods in Molecular Biology, 482, 281-294. https://doi.org/10.1007/978-1-59745-060-7_18
Griffiths-Jones, S., Saini, H. K., van Dongen, S., & Enright, A. J. (2007). miRBase: Tools for microRNA genomics. Nucleic Acids Research, 36, D154-D158. https://doi.org/10.1093/nar/gkm952
Hammond, S. M. (2015). An overview of microRNAs. Advanced Drug Delivery Reviews, 87, 3-14. https://doi.org/10.1016/j.addr.2015.05.001
Han, G., Zhang, L., Ni, X., Chen, Z., Pan, X., Zhu, Q., … Wang, X. (2018). MicroRNA-873 promotes cell proliferation, migration, and invasion by directly targeting TSLC1 in hepatocellular carcinoma. Cellular Physiology and Biochemistry, 46, 2261-2270. https://doi.org/10.1159/000489594
Inada, M., Yasui, T., Nomura, S., Miyake, S., Deguchi, K., Himeno, M., … Komori, T. (1999). Maturational disturbance of chondrocytes in Cbfa1-deficient mice. Developmental Dynamics, 214, 279-290. https://doi.org/10.1002/(SICI)1097-0177(199904)214
James, A. W. (2013). Review of signaling pathways governing MSC osteogenic and adipogenic differentiation. Scientifica, 2013, https://doi.org/10.1155/2013/684736
Jonason, J. H., Xiao, G., Zhang, M., Xing, L., & Chen, D. (2009). Post-translational regulation of Runx2 in bone and cartilage. Journal of Dental Research, 88(8), 693-703. https://doi.org/10.1177/0022034509341629
Kang, J. S., Alliston, T., Delston, R., & Derynck, R. (2005). Repression of Runx2 function by TGF-β through recruitment of class II histone deacetylases by Smad3. The EMBO Journal, 24, 2543-2555. https://doi.org/10.1038/sj.emboj.7600729
Kanoria, S., Rennie, W., Liu, C., Carmack, C. S., Lu, J., & Ding, Y. (2016). STarMir tools for prediction of microRNA binding sites, RNA structure determination. Methods in molecular biology (1490, pp. 73-82). New York, NY: Humana Press. https://doi.org/10.1007/978-1-4939-6433-8_6
Kapinas, K., & Delany, A. M. (2011). MicroRNA biogenesis and regulation of bone remodeling. Arthritis Research & Therapy, 13, 220. https://doi.org/10.1186/ar3325
Karsenty, G. (1998). Genetics of skeletogenesis. Developmental Genetics, 22(4), 301-313. https://doi.org/10.1002/(SICI)1520-6408(1998)22:4<301::AID-DVG1>3.0.CO;2-A
Ko, J. Y., Chuang, P. C., Ke, H. J., Chen, Y. S., Sun, Y. C., & Wang, F. S. (2015). MicroRNA-29a mitigates glucocorticoid induction of bone loss and fatty marrow by rescuing Runx2 acetylation. Bone, 81, 80-88. https://doi.org/10.1016/j.bone.2015.06.022
Komori, T. (2006). Regulation of osteoblast differentiation by transcription factors. Journal of Cellular Biochemistry, 99(5), 1233-1239. https://doi.org/10.1002/jcb.20958
Komori, T. (2017). Roles of Runx2 in skeletal development, RUNX Proteins in development and cancer. Advances in experimental medicine and biology (962, pp. 83-93). Singapore: Springer. https://doi.org/10.1007/978-981-10-3233-2_6
Lee, M., & Partridge, N. C. (2010). Parathyroid hormone activation of matrix metalloproteinase-13 transcription requires the histone acetyltransferase activity of p300 and PCAF and p300-dependent acetylation of PCAF. Journal of Biological Chemistry, 285(49), 38014-38022. https://doi.org/10.1074/jbc.M110.142141
Li, Z., Hassan, M. Q., Jafferji, M., Aqeilan, R. I., Garzon, R., Croce, C. M., … Lian, J. B. (2009). Biological functions of miR-29b contribute to positive regulation of osteoblast differentiation. Journal of Biological Chemistry, 284, 15676-15684. https://doi.org/10.1074/jbc.M809787200
Lian, J. B., Stein, G. S., Van Wijnen, A. J., Stein, J. L., Hassan, M. Q., Gaur, T., & Zhang, Y. (2012). MicroRNA control of bone formation and homeostasis. Nature Reviews Endocrinology, 8, 212-227. https://doi.org/10.1038/nrendo.2011.234
Liu, T. M., & Lee, E. H. (2012). Transcriptional regulatory cascades in Runx2-dependent bone development. Tissue Engineering Part B: Reviews, 19, 254-263. https://doi.org/10.1089/ten.teb.2012.0527
Lopes-Ramos, C. M., Paulson, J. N., Chen, C. Y., Kuijjer, M. L., Fagny, M., Platig, J., … Glass, K. (2017). Regulatory network changes between cell lines and their tissues of origin. BMC Genomics, 18(1), 723.
Malemud, C. J. (2006). Matrix metalloproteinases: Role in skeletal development and growth plate disorders. Frontiers in Bioscience, 11(11), 1702-1715.
McGee-Lawrence, M. E., & Westendorf, J. J. (2011). Histone deacetylases in skeletal development and bone mass maintenance. Gene, 474, 1-11. https://doi.org/10.1016/j.gene.2010.12.003
Mohanakrishnan, V., Balasubramanian, A., Mahalingam, G., Partridge, N. C., Ramachandran, I., & Selvamurugan, N. (2018). Parathyroid hormone-induced down-regulation of miR-532-5p for matrix metalloproteinase-13 expression in rat osteoblasts. Journal of Cellular Biochemistry, 119, 6181-6193. https://doi.org/10.1002/jcb.26827
Mokhlis, H. A., Bayraktar, R., Kabil, N. N., Caner, A., Kahraman, N., Rodriguez-Aguayo, C., … Ozpolat, B. (2019). The modulatory role of microRNA-873 in the progression of KRAS-driven cancers. Molecular Therapy-Nucleic Acids, 14, 301-317. https://doi.org/10.1016/j.omtn.2018.11.019
Moorthi, A., Vimalraj, S., Avani, C., He, Z., Partridge, N. C., & Selvamurugan, N. (2013). Expression of microRNA-30c and its target genes in human osteoblastic cells by nano-bioglass ceramic-treatment. International Journal of Biological Macromolecules, 56, 181-185. https://doi.org/10.1016/j.ijbiomac.2013.02.017
Nagase, H., & Woessner, J. F. (1999). Matrix metalloproteinases. Journal of Biological Chemistry, 274, 21491-21494. https://doi.org/10.1074/jbc.274.31.21491
Nakasa, T., Yoshizuka, M., Andry Usman, M., Elbadry Mahmoud, E., & Ochi, M. (2015). MicroRNAs and bone regeneration. Current Genomics, 16(6), 441-452. https://doi.org/10.2174/1389202916666150817213630
Nakatani, T., Chen, T., & Partridge, N. C. (2016). MMP-13 is one of the critical mediators of the effect of HDAC4 deletion on the skeleton. Bone, 90, 142-151. https://doi.org/10.1016/j.bone.2016.06.010
Narayanan, A., Srinaath, N., Rohini, M., & Selvamurugan, N. (2019). Regulation of Runx2 by microRNAs in osteoblast differentiation. Life Sciences, 232, 116676. https://doi.org/10.1016/j.lfs.2019.116676. 1. Epub 2019 Jul 21.
O'Hara, A. J., Vahrson, W., & Dittmer, D. P. (2008). Gene alteration and precursor and mature microRNA transcription changes contribute to the miRNA signature of primary effusion lymphoma. Blood, 111(4), 2347-2353. https://doi.org/10.1182/blood-2007-08-104463
Panaroni, C., Tzeng, Y. S., Saeed, H., & Wu, J. Y. (2014). Mesenchymal progenitors and the osteoblast lineage in bone marrow hematopoietic niches. Current Osteoporosis Reports, 12, 22-32. https://doi.org/10.1007/s11914-014-0190-7
Porte, D., Tuckermann, J., Becker, M., Baumann, B., Teurich, S., Higgins, T., … Angel, P. (1999). Both AP-1 and Cbfa1-like factors are required for the induction of interstitial collagenase by parathyroid hormone. Oncogene, 18(3), 667-678. https://doi.org/10.1038/sj.onc.1202333
Quinn, C. O., Scott, D. K., Brinckerhoff, C. E., Matrisian, L. M., Jeffrey, J. J., & Partridge, N. C. (1990). Rat collagenase. Cloning, amino acid sequence comparison, and parathyroid hormone regulation in osteoblastic cells. Journal of Biological Chemistry, 265(36), 22342-22347.
Rose, B. J., & Kooyman, D. L. (2016). A tale of two joints: The role of matrix metalloproteases in cartilage biology. Disease Markers, 2016, 1-7. https://doi.org/10.1155/2016/4895050
Saiganesh, S., Saathvika, R., Arumugam, B., Vishal, M., Udhaya, V., Ilangovan, R., & Selvamurugan, N. (2019). TGF-β1-stimulation of matrix metalloproteinase-13 expression by down-regulation of miR-203a-5p in rat osteoblasts. International Journal of Biological Macromolecules, 132, 541-549. https://doi.org/10.1016/j.ijbiomac.2019.04.003
Saiganesh, S., Saathvika, R., Udhaya, V., Arumugam, B., Vishal, M., & Selvamurugan, N. (2018). Matrix metalloproteinase-13: A special focus on its regulation by signaling cascades and microRNAs in bone. International Journal of Biological Macromolecules, 109, 338-349. https://doi.org/10.1016/j.ijbiomac.2017.12.091
Sampson, V. B., Yoo, S., Kumar, A., Vetter, N. S., & Kolb, E. A. (2015). MicroRNAs and potential targets in osteosarcoma. Frontiers in Pediatrics, 3, 69. https://doi.org/10.3389/fped.2015.00069
Schroeder, T. M., Jensen, E. D., & Westendorf, J. J. (2005). Runx2: A master organizer of gene transcription in developing and maturing osteoblasts. Birth Defects Research Part C: Embryo Today: Reviews, 75, 213-225. https://doi.org/10.1002/bdrc.20043
Selvamurugan, N., Chou, W. Y., Pearman, A. T., Pulumati, M. R., & Partridge, N. C. (1998). Parathyroid hormone regulates the rat collagenase-3 promoter in osteoblastic cells through the cooperative interaction of the activator protein-1 site and the runt domain binding sequence. Journal of Biological Chemistry, 273(17), 10647-10657. https://doi.org/10.1074/jbc.273.17.10647
Selvamurugan, N., Jefcoat, S. C., Kwok, S., Kowalewski, R., Tamasi, J. A., & Partridge, N. C. (2006). Overexpression of Runx2 directed by the matrix metalloproteinase-13 promoter containing the AP-1 and Runx/RD/Cbfa sites alters bone remodeling in vivo. Journal of Cellular Biochemistry, 99, 545-557. https://doi.org/10.1002/jcb.20878
Selvamurugan, N., Shimizu, E., Lee, M., Liu, T., Li, H., & Partridge, N. C. (2009). Identification and characterization of Runx2 phosphorylation sites involved in matrix metalloproteinase-13 promoter activation. FEBS Letters, 583, 1141-1146. https://doi.org/10.1016/j.febslet.2009.02.040
Shimizu, E., Nakatani, T., He, Z., & Partridge, N. C. (2014). Parathyroid hormone regulates histone deacetylase (HDAC) 4 through protein kinase A-mediated phosphorylation and dephosphorylation in osteoblastic cells. Journal of Biological Chemistry, 289, 21340-21350. https://doi.org/10.1074/sjbc.M114.550699
Shimizu, E., Selvamurugan, N., Westendorf, J. J., Olson, E. N., & Partridge, N. C. (2010). HDAC4 represses matrix metalloproteinase-13 transcription in osteoblastic cells and parathyroid hormone controls this repression. Journal of Biological Chemistry, 285, 9616-9626. https://doi.org/10.1074/jbc.M109.094862
Shimizu, E., Selvamurugan, N., Westendorf, J. J., & Partridge, N. C. (2007). Parathyroid hormone regulates histone deacetylases in osteoblasts. Annals of the New York Academy of Sciences, 1116(1), 349-353. https://doi.org/10.1196/annals.1402.037
Shreya, S., Malavika, D., Priya, V. R., & Selvamurugan, N. (2019). Regulation of histone deacetylases by microRNAs in bone. Current Protein and Peptide Science, 20, 356-367. https://doi.org/10.2174/1389203720666181031143129
Shui, C., Spelsberg, T. C., Riggs, B. L., & Khosla, S. (2003). Changes in Runx2/Cbfa1 expression and activity during osteoblastic differentiation of human bone marrow stromal cells. Journal of Bone and Mineral Research, 18, 213-221. https://doi.org/10.1359/jbmr.2003.18.2.213
Siddiqui, J. A., & Partridge, N. C. (2016). Physiological bone remodeling: Systemic regulation and growth factor involvement. Physiology, 31(3), 233-245. https://doi.org/10.1152/physiol.00061.2014
Smale, S. T. (2010). Luciferase assay. Cold Spring Harbor Protocols, 5, pdb-rot5421. https://doi.org/10.1101/pdb.prot5421
Stein, G. S., Lian, J. B., Van Wijnen, A. J., Stein, J. L., Montecino, M., Javed, A., & Pockwinse, S. M. (2004). Runx2 control of organization, assembly and activity of the regulatory machinery for skeletal gene expression. Oncogene, 23, 4315-4329. https://doi.org/10.1038/sj.onc.1207676
Vega, R. B., Matsuda, K., Oh, J., Barbosa, A. C., Yang, X., Meadows, E., … Olson, E. N. (2004). Histone deacetylase 4 controls chondrocyte hypertrophy during skeletogenesis. Cell, 119, 555-566. https://doi.org/10.1016/j.cell.2004.10.024
Vejnar, C. E., & Zdobnov, E. M. (2012). MiRmap: Comprehensive prediction of microRNA target repression strength. Nucleic Acids Research, 40, 11673-11683. https://doi.org/10.1093/nar/gks901
Vimalraj, S., Arumugam, B., Miranda, P. J., & Selvamurugan, N. (2015). Runx2: Structure, function, and phosphorylation in osteoblast differentiation. International Journal of Biological Macromolecules, 78, 202-208. https://doi.org/10.1016/j.ijbiomac.2015.04.008
Vimalraj, S., & Selvamurugan, N. (2012). MicroRNAs: Synthesis, gene regulation and osteoblast differentiation. Current Issues in Molecular Biology, 15(1), 7-18.
Vimalraj, S., & Selvamurugan, N. (2014). MicroRNAs expression and their regulatory networks during mesenchymal stem cells differentiation toward osteoblasts. International Journal of Biological Macromolecules, 66, 194-202. https://doi.org/10.1016/j.ijbiomac.2014.02.030
Vishal, M., Ajeetha, R., Keerthana, R., & Selvamurugan, N. (2016). Regulation of Runx2 by histone deacetylases in bone. Current Protein and Peptide Science, 17, 343-351. https://doi.org/10.2174/1389203716666150623104017
Vishal, M., Vimalraj, S., Ajeetha, R., Gokulnath, M., Keerthana, R., He, Z., … Selvamurugan, N. (2017). MicroRNA-590-5p stabilizes Runx2 by targeting Smad7 during osteoblast differentiation. Journal of Cellular Physiology, 232, 371-380. https://doi.org/10.1002/jcp.25434
Wang, X., Manner, P. A., Horner, A., Shum, L., Tuan, R. S., & Nuckolls, G. H. (2004). Regulation of MMP-13 expression by RUNX2 and FGF2 in osteoarthritic cartilage. Osteoarthritis and Cartilage, 12, 963-973. https://doi.org/10.1016/j.joca.2004.08.008
Wei, X., Li, H., Zhang, B., Li, C., Dong, D., Lan, X., … Chen, H. (2016). miR-378a-3p promotes differentiation and inhibits proliferation of myoblasts by targeting HDAC4 in skeletal muscle development. RNA Biology, 13, 1300-1309. https://doi.org/10.1080/15476286.2016.1239008
Westendorf, J. J. (2006). Transcriptional co-repressors of Runx2. Journal of Cellular Biochemistry, 98, 54-64. https://doi.org/10.1002/jcb.20805
Wibrand, K., Panja, D., Tiron, A., Ofte, M. L., Skaftnesmo, K. O., Lee, C. S., … Bramham, C. R. (2010). Differential regulation of mature and precursor microRNA expression by NMDA and metabotropic glutamate receptor activation during LTP in the adult dentate gyrus in vivo. European Journal of Neuroscience, 31, 636-645. https://doi.org/10.1111/j.1460-9568.2010.07112.x
Winchester, S. K., Selvamurugan, N., D'Alonzo, R. C., & Partridge, N. C. (2000). Developmental regulation of collagenase-3 mRNA in normal, differentiating osteoblasts through the activator protein-1 and therunt domain binding sites. Journal of Biological Chemistry, 275(30), 23310-23318. https://doi.org/10.1074/jbc.M003004200
Xing, T., Zhu, J., Xian, J., Li, A., Wang, X., Wang, W., & Zhang, Q. (2019). miRNA-548ah promotes the replication and expression of hepatitis B virus by targeting histone deacetylase 4. Life Sciences, 219, 199-208. https://doi.org/10.1016/j.lfs.2018.12.057
Zhang, C. (2010). Transcriptional regulation of bone formation by the osteoblast-specific transcription factor Osx. Journal of Orthopaedic Surgery and Research, 5, 37. https://doi.org/10.1186/1749-799X-5-37
Zhang, C., Tang, W., & Li, Y. (2012). Matrix metalloproteinase 13 (MMP13) is a direct target of osteoblast-specific transcription factor osterix (Osx) in osteoblasts. PLoS One, 7, e50525. https://doi.org/10.1371/journal.pone.0050525
Zhang, Y., Xie, R. L., Croce, C. M., Stein, J. L., Lian, J. B., Van Wijnen, A. J., & Stein, G. S. (2011). A program of microRNAs controls osteogenic lineage progression by targeting transcription factor Runx2. Proceedings of the National Academy of Sciences of the United States of America, 108(24), 9863-9868. https://doi.org/10.1073/pnas.1018493108
Zhang, Y., Xie, R. L., Gordon, J., LeBlanc, K., Stein, J. L., Lian, J. B., … Stein, G. S. (2012). Control of mesenchymal lineage progression by microRNAs targeting skeletal gene regulators Trps1 and Runx2. Journal of Biological Chemistry, 287, 21926-21935. https://doi.org/10.1074/jbc.M112.340398
Zhu, H., Guo, Z. K., Jiang, X. X., Li, H., Wang, X. Y., Yao, H. Y., … Mao, N. (2010). A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone. Nature Protocols, 5, 550-560. https://doi.org/10.1038/nprot.2009.238