信號傳導及轉錄激活蛋白3(Signal transducer and activator of transcription 3, STAT3) 屬於轉錄蛋白STAT家族的其中一員，受STAT3所調控之下游基因與細胞生長、分化、癌細胞轉移等特性息息相關；典型的激活模型指出當受到如上皮生長因子(EGFR)、介白質6 (Interlukin, IL-6) 等刺激時，STAT3會發生轉錄後修飾並聚合形成二聚體或是多聚體，進入細胞核與基因的啟動子區段結合，進而調控該基因表現；位於不同區段的轉錄後修飾會直接影響STAT3結構，並對STAT3的功能有所改變，此篇研究我們將討論酪胺酸705 (Tyrosine 705) 磷酸化、絲氨酸727 (Serine 727) 磷酸化、離胺酸685 (Lysine 685) 乙醯化在個別發生時對於肺癌癌幹細胞特性的調控，我們透過常間回文重複序列叢集/常間回文重複序列叢集關聯蛋白（Clustered Regularly Interspaced Short Palindromic Repeats/ CRISPR-Associated Protein 9, CRISPR/ Cas9）基因編輯系統將STAT3剔除，並轉染具有兩個位置或三個位置顯性抑制性突變的STAT3，並針對STAT3的入核情形以及下游所調控的基因作探討。實驗結果顯示無論在兩個位置或是三個位置被顯示抑制突變的情形下，細胞核的區域皆能偵測到STAT3的訊號，此結果證明了其他修飾點位對於STAT3入核也有所影響；而針對與EMT相關之Twist1調控，則發現在離胺酸685可被正常乙醯化的STAT3組別中，Twist1表現量與野生型(wild-type) STAT3並無顯著差異，顯示離胺酸685此點位對於STAT3調控Twist1的重要性。

Signal transducer and activator of transcription 3 (STAT3) is a well-known transcription factor in regulating genes related to cancer stemness. The canonical activation model of STAT3 suggests the importance of posttranslational modification (PTM), dimer formation, and nuclear translocation of STAT3 upon mediating the gene expression. Here, we aim to identify the independent contribution of three PTMs, including phosphorylation on tyrosine 705 (pY705) and serine 727 (pS727), and acetylation on lysine 685 (acetyl-K685), during STAT3 activation. First, STAT3-null lung cancer cells are generated by CRISPR-Cas9 knockout system, and expression of epithelial-mesenchymal transition (EMT)-related genes (N-cadherin, Twist) and stemness-related genes (Oct4, ABCB1) are found to be down-regulated after deletion of STAT3. Next, we introduce STAT3 mutants with double (Y705F/K685R, S727A/K685R, Y705F/S727A) or triple (Y705F/S727A/K685R) dominant-negative residue replacement. Although the capability of STAT3 nuclear translocation is not influenced when impairing S727, Y705, and K685 of STAT3, RT-PCR results show that acetyl-K685 STAT3 alone up-regulates the expression level of Twist1 equalling to the wild type STAT3. This study not only implies the potential of other PTMs in STAT3 dynamics but the specific role of acetyl-K685 in STAT3-dependent Twist1 regulation.

Contents
Abstract___________________________________________________________viii
Acknowledgement____________________________________________________x
Chapter 1. Introduction 1
1.1 Lung Cancer_____________________________________________________1
1.2 Signal Transducer and Activator of Transcription 3 (STAT3) 3
1.3 Cancer Stemness and STAT3 6
1.4 Purpose_________________________________________________________8
Chapter 2. Material and Methods 9
2.1 Cell Culture____________________________________________________ 9
2.2 Cell Counting__________________________________________________ 9
2.3 CRISPR/Cas9 Gene Editing System 10
2.3.1 STAT3 Knockout Plasmid 10
2.3.2 Liposome-Based Transfection - LipofectamineTM 2000 11
2.3.3 Puromycin Single Clone Selection 11
2.4 Electroporation_________________________________________________ 12
2.5 Cell Lysis_____________________________________________________ 12
2.6 Nuclear Cytosol Protein Fraction 13
2.7 Western Blot___________________________________________________ 14
2.8 RNA Isolation__________________________________________________ 14
2.9 RT-PCR_______________________________________________________ 15
2.10 Immunofluorescence (IF) 16
Chapter 3. Results____________________________________________________18
3.1 Characterization of STAT3 CRISPR/Cas9 Knockout HM20 cells 18
3.2 Down-regulation of ABCB1, Oct4, and Twist1 in STAT3 Knockout HM20 cells_________________ 19
3.3 Potential of other Non-canonical PTMs on STAT3 21
3.4 Acetyl-Lysine 685 is Essential for STAT3-dependent Twist1 Regulation 22
Chapter 4. Discussion 24
Chapter 5. Figures and Legends 29
Chapter 6. References 36
Chapter 7. Appendices 44

1. Herbst, R.S., D. Morgensztern, and C. Boshoff, The biology and management of non-small cell lung cancer. Nature, 2018. 553(7689): p. 446-454.
2. Zappa, C. and S.A. Mousa, Non-small cell lung cancer: current treatment and future advances. Transl Lung Cancer Res, 2016. 5(3): p. 288-300.
3. Duffy, M.J. and K. O'Byrne, Tissue and Blood Biomarkers in Lung Cancer: A Review. Adv Clin Chem, 2018. 86: p. 1-21.
4. Schrank, Z., et al., Current Molecular-Targeted Therapies in NSCLC and Their Mechanism of Resistance. Cancers (Basel), 2018. 10(7).
5. Jiao, X.D., et al., The prognostic value of TP53 and its correlation with EGFR mutation in advanced non-small cell lung cancer, an analysis based on cBioPortal data base. Lung Cancer, 2018. 123: p. 70-75.
6. Kempf, E., et al., 10-year long-term survival of a metastatic EGFR-mutated nonsmall cell lung cancer patient. Eur Respir J, 2015. 46(1): p. 280-2.
7. Yang, H., et al., New Horizons in KRAS-Mutant Lung Cancer: Dawn After Darkness. Front Oncol, 2019. 9: p. 953.
8. Lin, T. and Y. Lin, p53 switches off pluripotency on differentiation. Stem Cell Res Ther, 2017. 8(1): p. 44.
9. Testa, U., G. Castelli, and E. Pelosi, Lung Cancers: Molecular Characterization, Clonal Heterogeneity and Evolution, and Cancer Stem Cells. Cancers (Basel), 2018. 10(8).
10. Verhoeven, Y., et al., The potential and controversy of targeting STAT family members in cancer. Semin Cancer Biol, 2020. 60: p. 41-56.
11. Loh, C.Y., et al., Signal Transducer and Activator of Transcription (STATs) Proteins in Cancer and Inflammation: Functions and Therapeutic Implication. Front Oncol, 2019. 9: p. 48.
12. Sgrignani, J., et al., Structural Biology of STAT3 and Its Implications for Anticancer Therapies Development. Int J Mol Sci, 2018. 19(6).
13. Iwasaki, H., et al., Disruption of protein arginine N-methyltransferase 2 regulates leptin signaling and produces leanness in vivo through loss of STAT3 methylation. Circ Res, 2010. 107(8): p. 992-1001.
14. Dasgupta, M., et al., STAT3-driven transcription depends upon the dimethylation of K49 by EZH2. Proc Natl Acad Sci U S A, 2015. 112(13): p. 3985-90.
15. Hou, T., et al., The STAT3 NH2-terminal domain stabilizes enhanceosome assembly by interacting with the p300 bromodomain. J Biol Chem, 2008. 283(45): p. 30725-34.
16. Ray, S., et al., Requirement of histone deacetylase1 (HDAC1) in signal transducer and activator of transcription 3 (STAT3) nucleocytoplasmic distribution. Nucleic Acids Res, 2008. 36(13): p. 4510-20.
17. Icardi, L., et al., The Sin3a repressor complex is a master regulator of STAT transcriptional activity. Proc Natl Acad Sci U S A, 2012. 109(30): p. 12058-63.
18. Ray, S., et al., Inducible STAT3 NH2 terminal mono-ubiquitination promotes BRD4 complex formation to regulate apoptosis. Cell Signal, 2014. 26(7): p. 1445-55.
19. Yang, J., et al., Reversible methylation of promoter-bound STAT3 by histone-modifying enzymes. Proc Natl Acad Sci U S A, 2010. 107(50): p. 21499-504.
20. Kim, E., et al., Phosphorylation of EZH2 activates STAT3 signaling via STAT3 methylation and promotes tumorigenicity of glioblastoma stem-like cells. Cancer Cell, 2013. 23(6): p. 839-52.
21. Kim, J., et al., STAT3 regulation by S-nitrosylation: implication for inflammatory disease. Antioxid Redox Signal, 2014. 20(16): p. 2514-27.
22. Dasgupta, M., et al., Critical role for lysine 685 in gene expression mediated by transcription factor unphosphorylated STAT3. J Biol Chem, 2014. 289(44): p. 30763-71.
23. Lee, H., et al., Acetylated STAT3 is crucial for methylation of tumor-suppressor gene promoters and inhibition by resveratrol results in demethylation. Proc Natl Acad Sci U S A, 2012. 109(20): p. 7765-9.
24. Nie, Y., et al., STAT3 inhibition of gluconeogenesis is downregulated by SirT1. Nat Cell Biol, 2009. 11(4): p. 492-500.
25. Zouein, F.A., M. Kurdi, and G.W. Booz, Dancing rhinos in stilettos: The amazing saga of the genomic and nongenomic actions of STAT3 in the heart. JAKSTAT, 2013. 2(3): p. e24352.
26. Waitkus, M.S., et al., Signal integration and gene induction by a functionally distinct STAT3 phosphoform. Mol Cell Biol, 2014. 34(10): p. 1800-11.
27. Ng, I.H., M.A. Bogoyevitch, and D.A. Jans, Cytokine-induced slowing of STAT3 nuclear import; faster basal trafficking of the STAT3beta isoform. Traffic, 2014. 15(9): p. 946-60.
28. Gough, D.J., L. Koetz, and D.E. Levy, The MEK-ERK pathway is necessary for serine phosphorylation of mitochondrial STAT3 and Ras-mediated transformation. PLoS One, 2013. 8(11): p. e83395.
29. Tammineni, P., et al., The import of the transcription factor STAT3 into mitochondria depends on GRIM-19, a component of the electron transport chain. J Biol Chem, 2013. 288(7): p. 4723-32.
30. Zhang, Q., et al., Mitochondrial localized Stat3 promotes breast cancer growth via phosphorylation of serine 727. J Biol Chem, 2013. 288(43): p. 31280-8.
31. Wingelhofer, B., et al., Implications of STAT3 and STAT5 signaling on gene regulation and chromatin remodeling in hematopoietic cancer. Leukemia, 2018. 32(8): p. 1713-1726.
32. Zhou, Z., et al., SUMOylation and SENP3 regulate STAT3 activation in head and neck cancer. Oncogene, 2016. 35(45): p. 5826-5838.
33. Ng, J. and D. Cantrell, STAT3 is a serine kinase target in T lymphocytes. Interleukin 2 and T cell antigen receptor signals converge upon serine 727. J Biol Chem, 1997. 272(39): p. 24542-9.
34. Johnston, J.A., et al., Tyrosine phosphorylation and activation of STAT5, STAT3, and Janus kinases by interleukins 2 and 15. Proc Natl Acad Sci U S A, 1995. 92(19): p. 8705-9.
35. Billing, U., et al., Robustness and Information Transfer within IL-6-induced JAK/STAT Signalling. Commun Biol, 2019. 2: p. 27.
36. Fujita, R., et al., Anti-interleukin-6 receptor antibody (MR16-1) promotes muscle regeneration via modulation of gene expressions in infiltrated macrophages. Biochim Biophys Acta, 2014. 1840(10): p. 3170-80.
37. Hu, D., et al., Essential role of IL-10/STAT3 in chronic stress-induced immune suppression. Brain Behav Immun, 2014. 36: p. 118-27.
38. Rasmussen, T.K., et al., Overexpression of microRNA-155 increases IL-21 mediated STAT3 signaling and IL-21 production in systemic lupus erythematosus. Arthritis Res Ther, 2015. 17: p. 154.
39. Tang, Y., et al., STAT3 Genotypic Variant rs744166 and Increased Tyrosine Phosphorylation of STAT3 in IL-23 Responsive Innate Lymphoid Cells during Pathogenesis of Crohn's Disease. J Immunol Res, 2019. 2019: p. 9406146.
40. Floss, D.M., et al., Identification of canonical tyrosine-dependent and non-canonical tyrosine-independent STAT3 activation sites in the intracellular domain of the interleukin 23 receptor. J Biol Chem, 2013. 288(27): p. 19386-400.
41. Yokogami, K., et al., Serine phosphorylation and maximal activation of STAT3 during CNTF signaling is mediated by the rapamycin target mTOR. Curr Biol, 2000. 10(1): p. 47-50.
42. Wishingrad, M.A., S. Koshlukova, and S.W. Halvorsen, Ciliary neurotrophic factor stimulates the phosphorylation of two forms of STAT3 in chick ciliary ganglion neurons. J Biol Chem, 1997. 272(32): p. 19752-7.
43. Lelievre, E., et al., Signaling pathways recruited by the cardiotrophin-like cytokine/cytokine-like factor-1 composite cytokine: specific requirement of the membrane-bound form of ciliary neurotrophic factor receptor alpha component. J Biol Chem, 2001. 276(25): p. 22476-84.
44. Li, Y.J., et al., Crosstalk between ERK1/2 and STAT3 in the modulation of cardiomyocyte hypertrophy induced by cardiotrophin-1. Chin Med J (Engl), 2004. 117(8): p. 1135-42.
45. Huang, G., et al., STAT3 phosphorylation at tyrosine 705 and serine 727 differentially regulates mouse ESC fates. Stem Cells, 2014. 32(5): p. 1149-60.
46. Okada, Y., et al., Visualization and quantification of dynamic STAT3 homodimerization in living cells using homoFluoppi. Sci Rep, 2018. 8(1): p. 2385.
47. Ren, Z., et al., Phosphorylated STAT3 physically interacts with NPM and transcriptionally enhances its expression in cancer. Oncogene, 2015. 34(13): p. 1650-7.
48. White, U.A. and J.M. Stephens, The gp130 receptor cytokine family: regulators of adipocyte development and function. Curr Pharm Des, 2011. 17(4): p. 340-6.
49. Jackson, N.M. and B.P. Ceresa, EGFR-mediated apoptosis via STAT3. Exp Cell Res, 2017. 356(1): p. 93-103.
50. Blazevic, T., et al., 12/15-lipoxygenase contributes to platelet-derived growth factor-induced activation of signal transducer and activator of transcription 3. J Biol Chem, 2013. 288(49): p. 35592-603.
51. Deb, A., et al., Protein kinase PKR is required for platelet-derived growth factor signaling of c-fos gene expression via Erks and Stat3. EMBO J, 2001. 20(10): p. 2487-96.
52. Syed, Z.A., et al., HGF/c-met/Stat3 signaling during skin tumor cell invasion: indications for a positive feedback loop. BMC Cancer, 2011. 11: p. 180.
53. Gartsbein, M., et al., The role of protein kinase C delta activation and STAT3 Ser727 phosphorylation in insulin-induced keratinocyte proliferation. J Cell Sci, 2006. 119(Pt 3): p. 470-81.
54. Fu, A.K., et al., Cyclin-dependent kinase 5 phosphorylates signal transducer and activator of transcription 3 and regulates its transcriptional activity. Proc Natl Acad Sci U S A, 2004. 101(17): p. 6728-33.
55. Sakaguchi, M., et al., Role and regulation of STAT3 phosphorylation at Ser727 in melanocytes and melanoma cells. J Invest Dermatol, 2012. 132(7): p. 1877-85.
56. Kung, C.P., D.G. Meckes, Jr., and N. Raab-Traub, Epstein-Barr virus LMP1 activates EGFR, STAT3, and ERK through effects on PKCdelta. J Virol, 2011. 85(9): p. 4399-408.
57. Ohbayashi, N., et al., LIF- and IL-6-induced acetylation of STAT3 at Lys-685 through PI3K/Akt activation. Biol Pharm Bull, 2007. 30(10): p. 1860-4.
58. Wang, Y., et al., The role of STAT3 in leading the crosstalk between human cancers and the immune system. Cancer Lett, 2018. 415: p. 117-128.
59. Carpenter, R.L. and H.W. Lo, STAT3 Target Genes Relevant to Human Cancers. Cancers (Basel), 2014. 6(2): p. 897-925.
60. Vessoni, A.T., et al., Tumor propagating cells: drivers of tumor plasticity, heterogeneity, and recurrence. Oncogene, 2020. 39(10): p. 2055-2068.
61. Ravindran, S., S. Rasool, and C. Maccalli, The Cross Talk between Cancer Stem Cells/Cancer Initiating Cells and Tumor Microenvironment: The Missing Piece of the Puzzle for the Efficient Targeting of these Cells with Immunotherapy. Cancer Microenviron, 2019. 12(2-3): p. 133-148.
62. Qureshi-Baig, K., et al., Tumor-Initiating Cells: a criTICal review of isolation approaches and new challenges in targeting strategies. Mol Cancer, 2017. 16(1): p. 40.
63. Roma-Rodrigues, C., et al., Targeting Tumor Microenvironment for Cancer Therapy. Int J Mol Sci, 2019. 20(4).
64. Chen, F., et al., New horizons in tumor microenvironment biology: challenges and opportunities. BMC Med, 2015. 13: p. 45.
65. Balkwill, F.R., M. Capasso, and T. Hagemann, The tumor microenvironment at a glance. J Cell Sci, 2012. 125(Pt 23): p. 5591-6.
66. Hanahan, D. and R.A. Weinberg, Hallmarks of cancer: the next generation. Cell, 2011. 144(5): p. 646-74.
67. Grivennikov, S.I., F.R. Greten, and M. Karin, Immunity, inflammation, and cancer. Cell, 2010. 140(6): p. 883-99.
68. Yin, X., et al., Coexpression of gene Oct4 and Nanog initiates stem cell characteristics in hepatocellular carcinoma and promotes epithelial-mesenchymal transition through activation of Stat3/Snail signaling. J Hematol Oncol, 2015. 8: p. 23.
69. Won, C., et al., Signal transducer and activator of transcription 3-mediated CD133 up-regulation contributes to promotion of hepatocellular carcinoma. Hepatology, 2015. 62(4): p. 1160-73.
70. Jaggupilli, A. and E. Elkord, Significance of CD44 and CD24 as cancer stem cell markers: an enduring ambiguity. Clin Dev Immunol, 2012. 2012: p. 708036.
71. Li, B. and C. Huang, Regulation of EMT by STAT3 in gastrointestinal cancer (Review). Int J Oncol, 2017. 50(3): p. 753-767.
72. Singh, S. and R. Chakrabarti, Consequences of EMT-Driven Changes in the Immune Microenvironment of Breast Cancer and Therapeutic Response of Cancer Cells. J Clin Med, 2019. 8(5).
73. Roche, J., The Epithelial-to-Mesenchymal Transition in Cancer. Cancers (Basel), 2018. 10(2).
74. Pastushenko, I., et al., Identification of the tumour transition states occurring during EMT. Nature, 2018. 556(7702): p. 463-468.
75. Zhang, H.F. and R. Lai, STAT3 in Cancer-Friend or Foe? Cancers (Basel), 2014. 6(3): p. 1408-40.
76. Yang, M., et al., Expression profile and prognostic values of STAT family members in non-small cell lung cancer. Am J Transl Res, 2019. 11(8): p. 4866-4880.
77. Robey, R.W., et al., Revisiting the role of ABC transporters in multidrug-resistant cancer. Nat Rev Cancer, 2018. 18(7): p. 452-464.
78. Vesel, M., et al., ABCB1 and ABCG2 drug transporters are differentially expressed in non-small cell lung cancers (NSCLC) and expression is modified by cisplatin treatment via altered Wnt signaling. Respir Res, 2017. 18(1): p. 52.
79. Kim, S.J., et al., Alterations in Wnt- and/or STAT3 signaling pathways and the immune microenvironment during metastatic progression. Oncogene, 2019. 38(31): p. 5942-5958.
80. Jarzyna, P., N.V. Doan, and T.A. Deisher, Insertional mutagenesis and autoimmunity induced disease caused by human fetal and retroviral residual toxins in vaccines. Issues Law Med, 2016. 31(2): p. 221-234.
81. Chen, M.W., et al., The STAT3-miRNA-92-Wnt Signaling Pathway Regulates Spheroid Formation and Malignant Progression in Ovarian Cancer. Cancer Res, 2017. 77(8): p. 1955-1967.
82. Abell, A.N. and G.L. Johnson, Implications of Mesenchymal Cells in Cancer Stem Cell Populations: Relevance to EMT. Curr Pathobiol Rep, 2014. 2(1): p. 21-26.
83. Wu, S., et al., Upregulation of the EMT marker vimentin is associated with poor clinical outcome in acute myeloid leukemia. J Transl Med, 2018. 16(1): p. 170.
84. Saitoh, M., et al., STAT3 integrates cooperative Ras and TGF-beta signals that induce Snail expression. Oncogene, 2016. 35(8): p. 1049-57.
85. Xiong, H., et al., Roles of STAT3 and ZEB1 proteins in E-cadherin down-regulation and human colorectal cancer epithelial-mesenchymal transition. J Biol Chem, 2012. 287(8): p. 5819-32.
86. Cho, K.H., et al., STAT3 mediates TGF-beta1-induced TWIST1 expression and prostate cancer invasion. Cancer Lett, 2013. 336(1): p. 167-73.
87. Chiou, S.H., et al., Coexpression of Oct4 and Nanog enhances malignancy in lung adenocarcinoma by inducing cancer stem cell-like properties and epithelial-mesenchymal transdifferentiation. Cancer Res, 2010. 70(24): p. 10433-44.
88. Schaal, C.M., et al., Regulation of Sox2 and stemness by nicotine and electronic-cigarettes in non-small cell lung cancer. Mol Cancer, 2018. 17(1): p. 149.
89. Herreros-Pomares, A., et al., Lung tumorspheres reveal cancer stem cell-like properties and a score with prognostic impact in resected non-small-cell lung cancer. Cell Death Dis, 2019. 10(9): p. 660.
90. Miyakoshi, M., et al., Serine 727 phosphorylation of STAT3: an early change in mouse hepatocarcinogenesis induced by neonatal treatment with diethylnitrosamine. Mol Carcinog, 2014. 53(1): p. 67-76.
91. Ernst, S. and G. Muller-Newen, Nucleocytoplasmic Shuttling of STATs. A Target for Intervention? Cancers (Basel), 2019. 11(11).
92. Xu, L., et al., The STAT3 HIES mutation is a gain-of-function mutation that activates genes via AGG-element carrying promoters. Nucleic Acids Res, 2015. 43(18): p. 8898-912.
93. Vogt, M., et al., The role of the N-terminal domain in dimerization and nucleocytoplasmic shuttling of latent STAT3. J Cell Sci, 2011. 124(Pt 6): p. 900-9.
94. Liu, L., K.M. McBride, and N.C. Reich, STAT3 nuclear import is independent of tyrosine phosphorylation and mediated by importin-alpha3. Proc Natl Acad Sci U S A, 2005. 102(23): p. 8150-5.
95. Timofeeva, O.A., et al., Mechanisms of unphosphorylated STAT3 transcription factor binding to DNA. J Biol Chem, 2012. 287(17): p. 14192-200.
96. Koo, M.Y., et al., Selective inhibition of the function of tyrosine-phosphorylated STAT3 with a phosphorylation site-specific intrabody. Proc Natl Acad Sci U S A, 2014. 111(17): p. 6269-74.
97. Lee, S.C., et al., Essential role of insulin-like growth factor 2 in resistance to histone deacetylase inhibitors. Oncogene, 2016. 35(42): p. 5515-5526.
98. Min, H.Y., et al., Essential Role of DNA Methyltransferase 1-mediated Transcription of Insulin-like Growth Factor 2 in Resistance to Histone Deacetylase Inhibitors. Clin Cancer Res, 2017. 23(5): p. 1299-1311.
99. Li, J., et al., STAT3 acetylation-induced promoter methylation is associated with downregulation of the ARHI tumor-suppressor gene in ovarian cancer. Oncol Rep, 2013. 30(1): p. 165-70.
100. Sethi, G., et al., Inhibition of STAT3 dimerization and acetylation by garcinol suppresses the growth of human hepatocellular carcinoma in vitro and in vivo. Mol Cancer, 2014. 13: p. 66.
101. Sun, Y.P., et al., Genome-Wide Binding Studies of Acetyl-STAT3 Demonstrates a Novel Regulatory Pathway in Dendritic Cells. Blood, 2015. 126(23).
102. Xu, Y.S., et al., STAT3 Undergoes Acetylation-dependent Mitochondrial Translocation to Regulate Pyruvate Metabolism. Sci Rep, 2016. 6: p. 39517.