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 W. Zhou, et al. Cancer Letters 483 (2020) 75–86 In the present study, the expression and distribution of MICAL2 negative; 1, weak; 2, moderate; and 3, strong. PP was defined as fol- wereanalyzedinLUADtissuearrays,aswellasitsrelationshipwiththe lows: 0, negative; 1, 1–10% positive cells; 2, 11–50% positive cells; 3, clinicopathological characteristics of LUAD patients. Cell experiments 51–80% positive cells; and 4, more than 80% positive cells. Slices revealed tumor-promoting effects (growth, invasion, and EMT) invol- scoringnolessthan4pointswereclassifiedasimmunoreactive[24,25]. ving MICAL2 pathways in LUAD cell lines. Furthermore, our findings identifiedMICAL2asanucleocytoplasmicshuttlingprotein.Finally,the 2.4. Plasmids and constructs regulation and cytological function of MICAL2 nuclear export were investigated both in vitro and in vivo. The shMICAL2s were synthesized and purified by Hanyin Biotech Co. (Shanghai, China) and integrated into pHY-316 vector (Hanyin 2. Material

 W. Zhou, et al. Cancer Letters 483 (2020) 75–86 Fig. 1. Immunohistochemical analysis of MICAL2 expression in LUADs and paired normal lung tissues and its relationship to clinicopathological char- acteristics: A. MICAL2 expression levels are relatively lower in normal lung tissues and solely located in the nucleus, while it is highly expressed in LUADs and is mainlylocatedinthecytoplasm.TheMIofMICAL2inLUADsishighercomparedtopairednormallungtissues.Incontrasttopairednormallungtissues,mostLUADs have lower nuclear IRSs yet higher cytoplasmic IRSs. B. MICAL2 expression is higher in LUADs with lymphatic metastasis than in LUADs without lymphatic metastasis. Compared withnon-lymphatic-metastasis LUADs, lymphatic-metastasis LUADs have higher total and cytoplasmic MICAL2 expression levels, while there is no significant difference in nuclear MICAL2 levels. C. Total and cytoplasmic MICAL2 expression levels, but not nuclear MICAL2 expression levels, in LUAD are related to poor prognosis

 D. Zhu, et al. Cancer Letters 477 (2020) 107–121 Fig. 1. Dysbindin promotes PDAC metastasis and invasion in vitro and in vivo. (A) Western blot analysis showing dysbindin expression in PDAC and human pancreatic ductal epithelial cells. The data are presented as the mean ± SEM. (B) Western blotting and qRT-PCR analysis showing dysbindin expression in dysbindin-overexpressingcells(Aspc-1-LV-dysbindinandBxpc-3-LV-dysbindin)andcontrolcells(Aspc-1-LV-vectorandBxpc-3-LV-vector).Thedataarepresentedas themean ± SEM.(C)AfterPanc-1andCapan-2cellsweretransfectedwithLV-shDysbindin(sh1andsh2)orLV-shVector cck8 price ,WesternblottingandqRT-PCRanalysis wereperformed tomeasuredysbindinexpression.Thedataarepresentedasthe mean ± SEM.(D)OverexpressionofdysbindinenhancedAspc-1andBxpc-3cell migrationandinvasion.Thedataarepresentedasthemean ± SEM.(E)DysbindinknockdowndecreasedPanc-1andCapan-2cellmigrationandinvasion.Thedata are presented as the mean ± SEM. (F) In vivo metastasis assays.  Four

 W. Zhou, et al. Cancer Letters 483 (2020) 75–86 Fig. 3. MICAL2 shuttled between the cytoplasm and the nucleus: A. MICAL2 accumulated in the nucleus in PC-9 after treatment with LMB. The bottom panelshowsnuclearandcytoplasmicpositiveratesofendogenousMICAL2:thenuclearpositiveratedramaticallyincreasedafterLMBtreatment.B.Bothnuclearand cytoplasmiclocalizationofexogenousMICAL2inA549-MICAL2cellswithoutLMBtreatment Thymoquinone ,comparedtoastrongnuclearlocalizationwhentreatedwithLMB.The bottom panel shows that the MICAL2 cytoplasmic positive rate was significantly decreased after LMB treatment. C. MICAL2 locations in HeLa cells transfected with −ΔC WT and three truncated mutants of MICAL2. The right panel reveals a significant decrease in MICAL2 cytoplasmic positive rate in both MICAL2 and −ΔCΔN −ΔN MICAL2 transfected cells but not MICAL2 transfected cells compared with MICAL2-WT transfected cells. revealedinthecellexperiment,theirexpressionlevelsweredetermined LUAD metastasis. in

 C. Zhao, et al. Cancer Letters 481 (2020) 15–23 were calculated. for5min.Afterblockingwith5%bovineserumalbumin(BSA)/PBSfor 1 h, the cells were sequentially incubated with the primary antibodies 2.4. Annexin V/PI staining and Alexa Fluor-conjugated secondary (Abcam) at room temperature for 1 h. The cells were counterstained with DAPI for Apoptosis rates were determined by Annexin V-/PI double staining 10 min, and observed with a confocal laser microscope (Zeiss LSM510 as previously described[22]. Thesuitably treated cells wereharvested, Meta, Germany). washedwithabindingbuffer(Sigma-Aldrich),andthenincubatedwith 0.3μlAnnexinV-FITCandPIin100μlbindingbufferfor15mininthe 2.10. Xenograft model dark. The stained cells were observed under an inverted fluorescence microscope equipped with a digital camera (Axio Observer Z1, Zeiss, Nude BALB/c mice (male, 5-weeks old, weighing 18–22 g) were Germany). obtained from Guangdong Laboratory Animal Monitoring Institute (SCXK2008-2002), and

M.V. Yusenko, et al. Cancer Letters 479 (2020) 61–70 I B) genes, leading to disruption of the 3′-end of MYB mRNA, loss of Isolation of polyadenylated RNA and Northern blotting was performed MYB negative regulatory elements, and overexpression of oncogenic as described before [34]. Blots were hybridized sequentially to radi- MYB-NFIB fusion proteins [13]. MYB may also be activated by copy olabeled probes specific for MYB and ribosomal protein S17 mRNAs. numbergainorjuxtapositionofenhancerelementsfromNFIBandother Radioactive bands were quantified with a phosphor image analyzer. fusion partner genes [16,17]. Overall, thesefindings have greatly stimulated the interest in MYB 2.4. Flow cytometry as a target for drug development [1,3,18]. Although transcription fac- torsaretraditionally considereddifficulttotarget,initialapproaches to Approximately one million were cultured for 2 days in RPMI inhibit MYB activity or expression by small molecule or peptido-mi- 1640 medium containing 10

 Cancer Letters 479 (2020) 61–70 Contents lists available at ScienceDirect Cancer Letters journal homepage: www.elsevier.com/locate/canlet Original Articles Monensin, a novel potent MYB inhibitor, suppresses proliferation of acute T myeloid leukemia and adenoid cystic carcinoma cells a a b a a Maria V. Yusenko , Amke Trentmann ,Mattias K. Andersson ,Luca Abdel Ghani , Anke Jakobs , c c d b Mari-Francis Arteaga Paz , Jan-Henrik Mikesch , Jens Peter von Kries , Göran Stenman , a,∗ Karl-Heinz Klempnauer a Institute for Biochemistry, Westfälische-Wilhelms-Universität, D-48149, Münster, Germany b Sahlgrenska Cancer Center, Department of Pathology, University of Gothenburg, Gothenburg, Sweden c Department of Medicine A, Hematology and Oncology, University Hospital, Westfälische-Wilhelms-Universität, Münster, Germany d Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany ARTICLE INFO ABSTRACT Keywords: The master transcriptional regulator MYB is a key onc

 D. Zhu, et al. Cancer Letters 477 (2020) 107–121 Fig. 6. MiR-342–3p is downregulated in PDAC, and dysbindin is significantly negatively correlated with miR-342–3p. (A) MiR-342–3p is downregulated in PDAC according to The Cancer Genome Atlas (TCGA) data. NP, normal pancreas tissue; PC, pancreatic cancer tissue. (B) Dysbindin expression is upregulated in PDAC accordingtoTCGAdata.N,noncanceroustissue;T,tumourtissue.(C)qRT-PCRanalysisshowingmiR-342–3panddysbindinmRNAlevelsinPDACtissues(n=8) andpairedadjacentnoncanceroustissues.Thedataarepresentedasthemean ± SEM.(D)qRT-PCRanalysisshowingmiR-342–3pexpressionlevelsinPDACcell lines(Panc-1,Bxpc-3andAspc-1)andanormalpancreaticductalepithelialcellline(HPDE6c-7).Thedataarepresentedasthemean ± SEM.(E)Theassociation between dysbindin mRNA and miR-342–3p expression was analysed by Pearson\'s correlation coefficient (r = −0.6685, p < 0.01). (F) Kaplan-Meier survival analysis showing the overall survival of PDAC patients with high o

 D Zhu et al Cancer Letters Fig TheantitumoureffectofmiR ponPDACcelllinesisdiminishedbydysbindinoverexpression D. Zhu, et al. Cancer Letters 477 (2020) 107–121 Fig.7. TheantitumoureffectofmiR-342–3ponPDACcelllinesisdiminishedbydysbindinoverexpression,andmiR-342–3pdecreasesMDM2expression.(A)MiR- 342-3p-transfectedPDACcells(Aspc-1,Capan-2andPanc-1)werelessmetastaticandinvasivethancontrolcells.Thedataarepresentedasthemean ± SEM.(B) WesternblotanalysisshowingdysbindinproteinlevelsintheAspc-1-vector,Aspc-1-miR-342-mimicandAspc-1-miR-342-mimic+dysbindingroups.Thedataare presented as the mean ± SEM. (C) Transwell assays showing that dysbindin overexpression reverses the ability of miR-342–3p to inhibit PDAC cell (Aspc-1) migration and invasion. The data are presented as the mean ± SEM. (D) Western blot analysis showing MDM2 in miR-342-3p-transfected PDAC cells (Panc-1andBxpc-3)andcontrolcells.Thedataarepresentedasthemean ± SEM.(E)ProposedmodelbywhichtheNF-κB/MDM2signallingaxisisac

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