FGFR3/fibroblast growth factor receptor 3 inhibits autophagy through decreasing the ATG12–ATG5 conjugate, leading to the delay of cartilage development in achondroplasia

Autophagy

Volumn 11, Issue 11, 2015, Pages 1998-2013

  Wang, Xiaofeng ^a,b   Qi, Huabing ^a,b   Wang, Quan ^a,b   Zhu, Ying ^a,b   Wang, Xianxing ^a,b   Jin, Min ^a,b   Tan, Qiaoyan ^a,b   Huang, Qizhao ^a,b   Xu, Wei ^a,b   Li, Xiaogang ^a,b   Kuang, Liang ^a,b   Tang, Yubing ^a,b   Du, Xiaolan ^a,b   Chen, Di ^c   Chen, Lin ^a,b  

^a THIRD MILITARY MEDICAL UNIVERSITY   (China)

^b THIRD MILITARY MEDICAL UNIVERSITY   (China)

^c RUSH UNIVERSITY MEDICAL CENTER   (United States)

Author keywords

Achondroplasia; ATG12 ATG5 conjugate; Autophagy; Chondrocytes; FGFR3

Indexed keywords

AUTOPHAGY PROTEIN 12; AUTOPHAGY PROTEIN 5; CHLOROQUINE; FIBROBLAST GROWTH FACTOR RECEPTOR 3; PEPTIDES AND PROTEINS; UNCLASSIFIED DRUG;

ACHONDROPLASIA; ARTICLE; AUTOPHAGY; CARTILAGE; CARTILAGE CELL; CELL DIFFERENTIATION; CELL VIABILITY; CHONDROGENESIS; CONJUGATE; GROWTH RETARDATION; HUMAN; IN VITRO STUDY; IN VIVO STUDY; NONHUMAN; PATHOGENESIS; PROTEIN PROTEIN INTERACTION; TRANSIENT TRANSFECTION;

EID: 84964221939     PISSN: 15548627     EISSN: 15548635     Source Type: Journal    
DOI: 10.1080/15548627.2015.1091551     Document Type: Article

References (71)

1. Long F, Ornitz DM. Development of the endochondral skeleton. Cold Spring Harb Perspect Biol 2013; 5: a008334; PMID:23284041; http://dx.doi.org/ 10.1101/cshperspect.a008334
2. Erlebacher A, Filvaroff EH, Gitelman SE, Derynck R. Toward a molecular understanding of skeletal development. Cell 1995; 80:371-8; PMID:7859279; http://dx. doi.org/10.1016/0092-8674(95)90487-5
3. De Luca F, Baron J. Control of bone growth by fibroblast growth factors. Trends Endocrinol Metab 1999; 10:61-5; PMID:10322396; http://dx.doi.org/10.1016/ S1043-2760(98)00120-9
4. Bonaventure J, Rousseau F, Legeai-Mallet L, Le Merrer M, Munnich A, Maroteaux P. Common mutations in the gene encoding fibroblast growth factor receptor 3 account for achondroplasia, hypochondroplasia and thanatophoric dysplasia. Acta Paediatr Suppl 1996; 417:33-8; PMID:9055906; http://dx.doi.org/10.1111/j.1651-2227.1996. tb14291.x
5. Rousseau F, Bonaventure J, Legeai-Mallet L, Pelet A, Rozet JM, Maroteaux P, Le Merrer M, Munnich A. Mutations in the gene encoding fibroblast growth factor receptor-3 in achondroplasia. Nature 1994; 371:252-4; PMID:8078586; http://dx.doi.org/ 10.1038/371252a0
6. Vajo Z, Francomano CA, Wilkin DJ. The molecular and genetic basis of fibroblast growth factor receptor 3 disorders: the achondroplasia family of skeletal dysplasias, Muenke craniosynostosis, and Crouzon syndrome with acanthosis nigricans. Endocr Rev 2000; 21:23-39; PMID:10696568
7. Horton WA, Hall JG, Hecht JT. Achondroplasia. Lancet 2007; 370:162-72; PMID:17630040; http://dx.doi. org/10.1016/S0140-6736(07)61090-3
8. Briner J, Giedion A, Spycher MA. Variation of quantitative and qualitative changes of enchondral ossification in heterozygous achondroplasia. Pathol Res Pract 1991; 187:271-8; PMID:1906169; http://dx.doi.org/ 10.1016/S0344-0338(11)80783-1
9. Chen L, Adar R, Yang X, Monsonego EO, Li C, Hauschka PV, Yayon A, Deng CX. Gly369Cys mutation in mouse FGFR3 causes achondroplasia by affecting both chondrogenesis and osteogenesis. J Clin Invest 1999; 104:1517-25; PMID:10587515; http://dx.doi. org/10.1172/JCI6690
10. Deng C, Wynshaw-Boris A, Zhou F, Kuo A, Leder P. Fibroblast growth factor receptor 3 is a negative regulator of bone growth. Cell 1996; 84:911-21; PMID:8601314; http://dx.doi.org/10.1016/S0092-8674(00)81069-7
11. Chen L, Li C, Qiao W, Xu X, Deng C. A Ser(365)->Cys mutation of fibroblast growth factor receptor 3 in mouse downregulates Ihh/PTHrP signals and causes severe achondroplasia. Hum Mol Genet 2001; 10:457-65; PMID: 11181569; http://dx.doi.org/10.1093/ hmg/10.5.457
12. Yamanaka Y, Tanaka H, Koike M, Nishimura R, Seino Y. PTHrP rescues ATDC5 cells from apoptosis induced by FGF receptor 3 mutation. J Bone Miner Res 2003; 18:1395-403; PMID:12929929; http://dx. doi.org/10.1359/jbmr.2003.18.8.1395
13. Xie Y, Su N, Jin M, Qi H, Yang J, Li C, Du X, Luo F, Chen B, Shen Y, et al. Intermittent PTH (1-34) injection rescues the retarded skeletal development and postnatal lethality of mice mimicking human achondroplasia and thanatophoric dysplasia. Hum Mol Genet 2012; 21:3941-55; PMID:22634226; http://dx. doi.org/10.1093/hmg/dds181
14. Naski MC, Colvin JS, Coffin JD, Ornitz DM. Repression of hedgehog signaling and BMP4 expression in growth plate cartilage by fibroblast growth factor receptor 3. Development 1998; 125:4977-88; PMID:9811582
15. Qi H, Jin M, Duan Y, Du X, Zhang Y, Ren F,Wang Y, Tian Q, Wang X, Wang Q, et al. FGFR3 induces degradation of BMP type I receptor to regulate skeletal development. Biochim Biophys Acta 2014; 1843:1237-47; PMID:24657641; http://dx.doi.org/10.1016/j. bbamcr.2014.03.011
16. Rothenbuhler A, Linglart A, Piquard C, Bougneres P. A pilot study of discontinuous, insulin-like growth factor 1-dosing growth hormone treatment in young children with FGFR3 N540K-mutated hypochondroplasia. J Pediatr 2012; 160:849-53; PMID:22137367; http://dx.doi.org/ 10.1016/j.jpeds.2011.10.023
17. Jin M, Klionsky DJ. Regulation of autophagy: Modulation of the size and number of autophagosomes. FEBS Lett 2014; 588:2457-63; PMID:24928445; http://dx. doi.org/10.1016/j.febslet.2014.06.015
18. Lotz MK, Carames B. Autophagy and cartilage homeostasis mechanisms in joint health, aging and OA. Nat Rev Rheumatol 2011; 7:579-87; PMID:21808292
19. Bohensky J, Shapiro IM, Leshinsky S, Watanabe H, Srinivas V. PIM-2 is an independent regulator of chondrocyte survival and autophagy in the epiphyseal growth plate. J Cell Physiol 2007; 213:246-51; PMID:17476689; http:// dx.doi.org/10.1002/jcp.21117
20. Bohensky J, Shapiro IM, Leshinsky S, Terkhorn SP, Adams CS, Srinivas V. HIF-1 regulation of chondrocyte apoptosis: induction of the autophagic pathway. Autophagy 2007; 3:207-14; PMID:17224629; http:// dx.doi.org/10.4161/auto.3708
21. Bohensky J, Leshinsky S, Srinivas V, Shapiro IM. Chondrocyte autophagy is stimulated by HIF-1 dependent AMPK activation and mTOR suppression. Pediatr Nephrol 2010; 25:633-42; PMID:19830459; http://dx.doi.org/10.1007/s00467-009-1310-y
22. Shapiro IM, Adams CS, Freeman T, Srinivas V. Fate of the hypertrophic chondrocyte: microenvironmental perspectives on apoptosis and survival in the epiphyseal growth plate. Birth Defects Res C Embryo Today 2005; 75:330-9; PMID:16425255; http://dx.doi.org/ 10.1002/bdrc.20057
23. Bohensky J, Terkhorn SP, Freeman TA, Adams CS, Garcia JA, Shapiro IM, Srinivas V. Regulation of autophagy in human and murine cartilage: hypoxiainducible factor 2 suppresses chondrocyte autophagy. Arthritis Rheum 2009; 60:1406-15; PMID:19404942; http://dx.doi.org/10.1002/art.24444
24. Carames B, Taniguchi N, Otsuki S, Blanco FJ, Lotz M. Autophagy is a protective mechanism in normal cartilage, and its aging-related loss is linked with cell death and osteoarthritis. Arthritis Rheum 2010; 62:791-801; PMID:20187128; http://dx.doi.org/10.1002/art.27305
25. Carames B, Taniguchi N, Seino D, Blanco FJ, D'Lima D, Lotz M. Mechanical injury suppresses autophagy regulators and pharmacologic activation of autophagy results in chondroprotection. Arthritis Rheum 2012; 64:1182-92; PMID:22034068; http://dx.doi.org/ 10.1002/art.33444
26. Sasaki H, Takayama K, Matsushita T, Ishida K, Kubo S, Matsumoto T, Fujita N, Oka S, Kurosaka M, Kuroda R. Autophagy modulates osteoarthritis-related gene expression in human chondrocytes. Arthritis Rheum 2012; 64:1920-8; PMID:22147463; http://dx.doi.org/ 10.1002/art.34323
27. Zhang M, Zhang J, Lu L, Qiu ZY, Zhang X, Yu SB, Wu YP, Wang MQ. Enhancement of chondrocyte autophagy is an early response in the degenerative cartilage of the temporomandibular joint to biomechanical dental stimulation. Apoptosis 2013; 18:423-34; PMID:23386193; http://dx.doi.org/10.1007/s10495-013-0811-0
28. Settembre C, Arteaga-Solis E, McKee MD, de Pablo R, Al Awqati Q, Ballabio A, Karsenty G. Proteoglycan desulfation determines the efficiency of chondrocyte autophagy and the extent of FGF signaling during endochondral ossification. Genes Dev 2008; 22:2645-50; PMID:18832069; http://dx.doi.org/10.1101/ gad.1711308
29. Hall-Glenn F, Aivazi A, Akopyan L, Ong JR, Baxter RR, Benya PD, Goldschmeding R, van Nieuwenhoven FA, Hunziker EB, Lyons KM. CCN2/CTGF is required for matrix organization and to protect growth plate chondrocytes from cellular stress. J Cell Commun Signal 2013; 7:219-30; PMID:23666466; http://dx. doi.org/10.1007/s12079-013-0201-y
30. Lin X, Zhang Y, Liu L, McKeehan WL, Shen Y, Song S, Wang F. FRS2alpha is essential for the fibroblast growth factor to regulate the mTOR pathway and autophagy in mouse embryonic fibroblasts. Int J Biol Sci 2011; 7:1114-21; PMID:21927580; http://dx.doi. org/10.7150/ijbs.7.1114
31. Zhang J, Liu J, Huang Y, Chang JY, Liu L, McKeehan WL, Martin JF, Wang F. FRS2alpha-mediated FGF signals suppress premature differentiation of cardiac stem cells through regulating autophagy activity. Circ Res 2012; 110:e29-39; PMID:22207710; http://dx. doi.org/10.1161/CIRCRESAHA.111.255950
32. Srinivas V, Bohensky J, Shapiro IM. Autophagy: a new phase in the maturation of growth plate chondrocytes is regulated by HIF, mTOR and AMP kinase. Cells Tissues Organs 2009; 189:88-92; PMID:18703865; http://dx.doi.org/10.1159/000151428
33. Srinivas V, Shapiro IM. Chondrocytes embedded in the epiphyseal growth plates of long bones undergo autophagy prior to the induction of osteogenesis. Autophagy 2006; 2:215-6; PMID:16874108; http:// dx.doi.org/10.4161/auto.2649
34. Su N, Xu X, Li C, He Q, Zhao L, Li C, Chen S, Luo F, Yi L, Du X, et al. Generation of Fgfr3 conditional knockout mice. Int J Biol Sci 2010; 6:327-32; PMID:20582225; http://dx.doi.org/10.7150/ijbs.6.327
35. Klionsky DJ, Abdalla FC, Abeliovich H, Abraham RT, Acevedo-Arozena A, Adeli K, Agholme L, Agnello M, Agostinis P, Aguirre-Ghiso JA, et al. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 2012; 8:445-544; PMID:22966490; http://dx.doi.org/10.4161/auto.19496
36. Mukhopadhyay K, Lefebvre V, Zhou G, Garofalo S, Kimura JH, de Crombrugghe B. Use of a new rat chondrosarcoma cell line to delineate a 119-base pair chondrocyte-specific enhancer element and to define active promoter segments in the mouse pro-alpha 1(II) collagen gene. J Biol Chem 1995; 270:27711-9; PMID:7499238; http://dx.doi.org/10.1074/ jbc.270.6.2827
37. Krejci P, Salazar L, Kashiwada TA, Chlebova K, Salasova A, Thompson LM, Bryja V, Kozubik A, Wilcox WR. Analysis of STAT1 activation by six FGFR3 mutants associated with skeletal dysplasia undermines dominant role of STAT1 in FGFR3 signaling in cartilage. PLoS One 2008; 3:e3961; PMID:19088846; http://dx.doi.org/10.1371/journal.pone.0003961
38. Sahni M, Ambrosetti DC, Mansukhani A, Gertner R, Levy D, Basilico C. FGF signaling inhibits chondrocyte proliferation and regulates bone development through the STAT-1 pathway. Genes Dev 1999; 13:1361-6; PMID:10364154; http://dx.doi.org/10.1101/ gad.13.11.1361
39. Krejci P, Masri B, Salazar L, Farrington-Rock C, Prats H, Thompson LM, Wilcox WR. Bisindolylmaleimide I suppresses fibroblast growth factor-mediated activation of Erk MAP kinase in chondrocytes by preventing Shp2 association with the Frs2 and Gab1 adaptor proteins. J Biol Chem 2007; 282:2929-36; PMID:17145761; http://dx.doi.org/10.1074/jbc. M606144200
40. Altaf FM, Hering TM, Kazmi NH, Yoo JU, Johnstone B. Ascorbate-enhanced chondrogenesis of ATDC5 cells. Eur Cell Mater 2006; 12:64-9; discussion 9-70
41. Jin M, Yu Y, Qi H, Xie Y, Su N, Wang X, Tan Q, Luo F, Zhu Y, Wang Q, et al. A novel FGFR3-binding peptide inhibits FGFR3 signaling and reverses the lethal phenotype of mice mimicking human thanatophoric dysplasia. Hum Mol Genet 2012; 21:5443-55; PMID:23014564; http://dx.doi.org/10.1093/hmg/ dds390
42. Gao C, Cao W, Bao L, Zuo W, Xie G, Cai T, Fu W, Zhang J, Wu W, Zhang X, et al. Autophagy negatively regulates Wnt signalling by promoting Dishevelled degradation. Nat Cell Biol 2010; 12:781-90; PMID:20639871; http://dx.doi.org/10.1038/ncb2082
43. Yang J, Carra S, Zhu WG, Kampinga HH. The regulation of the autophagic network and its implications for human disease. Int J Biol Sci 2013; 9:1121-33; PMID:24339733; http://dx.doi.org/10.7150/ijbs.6666
44. Mizushima N, Noda T, Yoshimori T, Tanaka Y, Ishii T, George MD, Klionsky DJ, Ohsumi M, Ohsumi Y. A protein conjugation system essential for autophagy. Nature 1998; 395:395-8; PMID:9759731; http://dx. doi.org/10.1038/26506
45. Mizushima N, Yamamoto A, Hatano M, Kobayashi Y, Kabeya Y, Suzuki K, Tokuhisa T, Ohsumi Y, Yoshimori T. Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells. J Cell Biol 2001; 152:657-68; PMID:11266458; http://dx.doi. org/10.1083/jcb.152.4.657
46. Kuma A, Hatano M, Matsui M, Yamamoto A, Nakaya H, Yoshimori T, Ohsumi Y, Tokuhisa T, Mizushima N. The role of autophagy during the early neonatal starvation period. Nature 2004; 432:1032-6; PMID:15525940; http://dx.doi.org/10.1038/nature 03029
47. Ewing RM, Chu P, Elisma F, Li H, Taylor P, Climie S, McBroom-Cerajewski L, Robinson MD, O'Connor L, Li M, et al. Large-scale mapping of human proteinprotein interactions by mass spectrometry. Mol Syst Biol 2007; 3:89; PMID:17353931; http://dx.doi.org/ 10.1038/msb4100134
48. Remy I, Montmarquette A, Michnick SW. PKB/Akt modulates TGF-beta signalling through a direct interaction with Smad3. Nat Cell Biol 2004; 6:358-65; PMID:15048128; http://dx.doi.org/10.1038/ncb1113
49. Qiu T, Wu X, Zhang F, Clemens TL, Wan M, Cao X. TGF-beta type II receptor phosphorylates PTH receptor to integrate bone remodelling signalling. Nat Cell Biol 2010; 12:224-34; PMID:20139972
50. Pyo JO, Jang MH, Kwon YK, Lee HJ, Jun JI, Woo HN, Cho DH, Choi B, Lee H, Kim JH, et al. Essential roles of Atg5 and FADD in autophagic cell death: dissection of autophagic cell death into vacuole formation and cell death. J Biol Chem 2005; 280:20722-9; PMID:15778222; http://dx.doi.org/10.1074/jbc. M413934200
51. Pyo JO, Yoo SM, Ahn HH, Nah J, Hong SH, Kam TI, Jung S, Jung YK. Overexpression of Atg5 in mice activates autophagy and extends lifespan. Nat Commun 2013; 4:2300; PMID:23939249; http://dx.doi.org/ 10.1038/ncomms3300
52. Krejci P, Prochazkova J, Smutny J, Chlebova K, Lin P, Aklian A, Bryja V, Kozubik A, Wilcox WR. FGFR3 signaling induces a reversible senescence phenotype in chondrocytes similar to oncogene-induced premature senescence. Bone 2010; 47:102-10; PMID:20362703; http://dx.doi.org/10.1016/j.bone.2010.03.021
53. Foldynova-Trantirkova S, Wilcox WR, Krejci P. Sixteen years and counting: the current understanding of fibroblast growth factor receptor 3 (FGFR3) signaling in skeletal dysplasias. Hum Mutat 2012; 33:29-41; PMID:22045636; http://dx.doi.org/10.1002/humu. 21636
54. Yasoda A, Kitamura H, Fujii T, Kondo E, Murao N, Miura M, Kanamoto N, Komatsu Y, Arai H, Nakao K. Systemic administration of C-type natriuretic peptide as a novel therapeutic strategy for skeletal dysplasias. Endocrinology 2009; 150:3138-44; PMID:19282381; http://dx.doi.org/10.1210/en.2008-1676
55. Lorget F, Kaci N, Peng J, Benoist-Lasselin C, Mugniery E, Oppeneer T, Wendt DJ, Bell SM, Bullens S, Bunting S, et al. Evaluation of the therapeutic potential of a CNP analog in a Fgfr3 mouse model recapitulating achondroplasia. Am J Hum Genet 2012; 91:1108-14; PMID:23200862; http://dx.doi.org/10.1016/j. ajhg.2012.10.014
56. Garcia S, Dirat B, Tognacci T, Rochet N, Mouska X, Bonnafous S, Patouraux S, Tran A, Gual P, Le Marchand-Brustel Y, et al. Postnatal soluble FGFR3 therapy rescues achondroplasia symptoms and restores bone growth in mice. Sci Transl Med 2013; 5:203ra124; PMID:24048522
57. Yamashita A, Morioka M, Kishi H, Kimura T, Yahara Y, Okada M, Fujita K, Sawai H, Ikegawa S, Tsumaki N. Statin treatment rescues FGFR3 skeletal dysplasia phenotypes. Nature 2014; 513:507-11; PMID:25231866; http://dx.doi.org/10.1038/nature 13775
58. Cecconi F, Levine B. The role of autophagy in mammalian development: cell makeover rather than cell death. Dev cell 2008; 15:344-57; PMID:18804433; http://dx.doi.org/10.1016/j.devcel.2008.08.012
59. Settembre C, Arteaga-Solis E, Ballabio A, Karsenty G. Self-eating in skeletal development: implications for lysosomal storage disorders. Autophagy 2009; 5:228-9; PMID:19029806; http://dx.doi.org/10.4161/ auto.5.2.7390
60. Du X, Xie Y, Xian CJ, Chen L. Role of FGFs/FGFRs in skeletal development and bone regeneration. J Cell Physiol 2012; 227:3731-43; PMID:22378383; http:// dx.doi.org/10.1002/jcp.24083
61. Su N, Jin M, Chen L. Role of FGF/FGFR signaling in skeletal development and homeostasis: learning from mouse models. Bone Res 2014; 2:14003; PMID:26273516; http://dx.doi.org/10.1038/boneres. 2014.3
62. Kuma A, Mizushima N, Ishihara N, Ohsumi Y. Formation of the approximately 350-kDa Apg12-Apg5. Apg16 multimeric complex, mediated by Apg16 oligomerization, is essential for autophagy in yeast. J Biol Chem 2002; 277:18619-25; PMID:11897782; http:// dx.doi.org/10.1074/jbc.M111889200
63. Mizushima N, Kuma A, Kobayashi Y, Yamamoto A, Matsubae M, Takao T, Natsume T, Ohsumi Y, Yoshimori T. Mouse Apg16L, a novel WD-repeat protein, targets to the autophagic isolation membrane with the Apg12-Apg5 conjugate. J Cell Sci 2003; 116:1679-88; PMID:12665549; http://dx.doi.org/10.1242/jcs.00381
64. Hanada T, Noda NN, Satomi Y, Ichimura Y, Fujioka Y, Takao T, Inagaki F, Ohsumi Y. The Atg12-Atg5 conjugate has a novel E3-like activity for protein lipidation in autophagy. J Biol Chem 2007; 282:37298-302; PMID:17986448; http://dx.doi.org/10.1074/jbc. C700195200
65. Bell BD, Leverrier S, Weist BM, Newton RH, Arechiga AF, Luhrs KA, Morrissette NS,Walsh CM. FADD and caspase-8 control the outcome of autophagic signaling in proliferating T cells. Proc Natl Acad Sci U S A 2008; 105:16677-82; PMID:18946037; http://dx.doi. org/10.1073/pnas.0808597105
66. Chen ZH, Cao JF, Zhou JS, Liu H, Che LQ, Mizumura K, LiW, Choi AM, Shen HH. Interaction of caveolin-1 with ATG12-ATG5 system suppresses autophagy in lung epithelial cells. Am J Physiol Lung Cell Mol Physiol 2014; 306:L1016-25; PMID:24727585; http://dx.doi.org/10.1152/ajplung. 00268.2013
67. Ogawa M, Yoshimori T, Suzuki T, Sagara H, Mizushima N, Sasakawa C. Escape of intracellular Shigella from autophagy. Science 2005; 307:727-31; PMID:15576571; http://dx.doi.org/10.1126/science. 1106036
68. Gosset M, Berenbaum F, Thirion S, Jacques C. Primary culture and phenotyping of murine chondrocytes. Nat Protoc 2008; 3:1253-60; PMID:18714293; http:// dx.doi.org/10.1038/nprot.2008.95
69. Saiki S, Sasazawa Y, Imamichi Y, Kawajiri S, Fujimaki T, Tanida I, Kobayashi H, Sato F, Sato S, Ishikawa K, et al. Caffeine induces apoptosis by enhancement of autophagy via PI3K/Akt/mTOR/p70S6K inhibition. Autophagy 2011; 7:176-87; PMID:21081844; http:// dx.doi.org/10.4161/auto.7.2.14074
70. Sarkar S, Davies JE, Huang Z, Tunnacliffe A, Rubinsztein DC. Trehalose, a novel mTOR-independent autophagy enhancer, accelerates the clearance of mutant huntingtin and alpha-synuclein. J Biol Chem 2007; 282:5641-52; PMID:17182613; http://dx.doi.org/ 10.1074/jbc.M609532200
71. Strober W. Monitoring cell growth. Curr Protoc Immunol. 2001; 21:3A:A.3A.1-A.3A.2; PMID: 18432653; http://dx.doi.org/10.1002/0471142735. ima03as21