Cell Counting Kit-8 mw

      X.Zhang,etal. Cancer Letters 481 (2020) 32–44 2.10. Immunofluorescencemicroscopy immunofluorescence and validated the co-localization of SSL6 and CD47 on the MHCC97H cell membrane and the down-regulated CD47 4 MHCC97H cells were inoculated into a 24-well plate at 1 × 10 / by SSL6 treatment (24 h: the reduction percentage of SSL6 vs. control well. Upon the cell adherence, they were fixed with 4% paraf- was 3.44%, p = 0.4636 72 h: the reduction percentage of SSL6 vs. ormaldehyde. Cells were stained with Anti-CD47 antibody (ab175388, control was 22.87%, p = 0.0036) (Fig. 2C and D). When SSL6 was not Acbam, Cambridge, UK) and Anti-6X His-tag antibody (ab18184, administrated, the His-tag was stained in the nuclei (Fig. 2C and D) as Abcam)overnightat4°C.Thencellswerewashedfor3timeswithPBS reported previously [33].    Consistent with the data from immuno- and then were incubated for 30 min with secondary Alexa- fluorescence, the protein expression of CD47 was not changed af

  M.V. Yusenko, et al. Cancer Letters 479 (2020) 61–70 set of genes down regulated by MYB-NFIB knockdown in ACC cells [23] MF498 . We observed a significant overlap between genes suppressed by monensin in THP1 and genes activated byMYB-NFIB in ACCcells, indicating that monensin targets MYB-NFIB-dependent gene transcrip- tion in ACC cells (Fig. 7E). qPCR analysis of the effects of monensin on theexpressionofselectedgenesinACC1cellssupportedthisconclusion (Fig cck8 ic50 . 7F). 4. Discussion MYB has emerged as a candidate drug target for human malig- nancies whose development depends on deregulated MYB expression, such as AML and ACC. Although targeting transcription factors has Fig. 6. Effect of monensin on clonal growth of primary murine AML cells traditionally been considered difficult, recent work from our and other and normal hematopoietic progenitors. Kit positive cells from bone marrow of healthy mice and murine AML cells carrying the MLL-AF9 on cofusion groups has demonstra

  r> X.Zhang,etal. Cancer Letters 481 (2020) 32–44 Fig.1. SSL6promotesSFN-inducedapoptosis ofHCCcells.(A)Theprotocolfor SSL6synthesis.(B)Western-blot identificationoftheprokaryoticSSL6proteinwith His-tag.(C)ProteinelectrophoresisofSSL6.(D,E)Flowcytometryofcelldeath(AnnexinVand7AAD)wasperformedwithHuh-7(D)andMHCC97H(E)cellstreated with indicated doses of SFN and/or SSL6. Data are expressed as means ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. or SSL6 for indicated intervals, each sample was prepared as a 100 μL USA). In some experiments, PI3K inhibitor LY294002 (S1737, single cell suspension using pre-cooled PBS for staining. The samples Beyotime) and the inhibitor of glycolysis 2-DG (D8375,Sigma-Aldrich) were stained with FITC anti-human CD47 (323106, Biolegend, MA, were administrated. USA) and FITC Mouse IgG1, κ Isotype (400109, Biolegend) in the dark for30minat4°Cfollowingthemanufacturers\'protocols.ForAnnexin- 2.8. Lactateassay V/7AAD staining, cel

  C. Zhao, et al. Cancer Letters 481 (2020) 15–23 Fig. 1. Prodigiosin induces autop- hagic alterations in human colon cancer cells. (A) The chemical structures of prodi- giosin (PG). (B and C) Prodigiosin pro- motes LC3B-II accumulation. The cells were exposed to prodigiosin at the dosesindicatedfor12h.(B),ortreated with prodigiosin (0.2 μM) for the time periods indicated. (C), and cell lysates were analyzed using western blotting along with an anti-LC3B antibody. (D) Prodigiosin induces LC3B-II accumula- tion in multiple colon cancer cell lines. Three different lines of colon cancer cells were treated with prodigiosin (0.2 μM) for 12 h and the cells lysates were analyzed using western blotting. (E and F) Effect of prodigiosin on GFP- LC3B punctation. SW480 cells stably expressingEGFP-LC3weretreatedwith various concentrations of prodigiosin for 12 h. (E) or were treated with 0.2 μM prodigiosin for the different durations indicated (F), and the EGFP- LC3 puncta were observed u

  W. Zhou, et al. Cancer Letters 483 (2020) 75–86 -ΔC −ΔC Fig.5. ComparedtoMICAL2,MICAL2 hadlesspromotingeffectonproliferationandmobilityinLUADcells in vitroandmetastasis in vivo:A.MICAL2 −ΔC andMICAL2wereexpressedintransfectedA549cells.Myosin-9wasenhancedinbothA549-MICAL2 andA549-MICAL2cellscomparedwithA549-CTRLcells.B. −ΔC A549-MICAL2 showed a significant increase in proliferation compared with A549-CTRL, while at a significantly lower level compared with A549-MICAL2 cells. −ΔC C. The cell migration (top) and invasion (bottom) assays indicated that MICAL2 did not promote migration or invasion in A549 as in MICAL2-WT cells. D.    Representativeimagesoflungmetastaticnodules(circlesmarkedwithpartoftumormetastaticfocus)derivedfromA549cellswithorwithoutstablyoverexpressing ΔC −ΔC MICAL2- or MICAL2. A549-MICAL2 showed a significant increase in metastatic nodule number while A549-MICAL2 did not (n = 4, *** = P < 0 Mitomycin C .001). In addition, several articles report that myosi

 M.V. Yusenko, et al. Cancer Letters 479 (2020) 61–70 Fig. 1. Identification of monensin as a potent MYB inhibitor. A. Schematic illus- tration of the HEK-MYB-Luc reporter cell line. The carry a stably integrated MYB-inducible luciferase reporter gene, an expression vector for human MYB-2KR under the control of a modified CMV pro- moter with Tet-operator sites positioned close to the transcriptional start site and an expression vector for the Tet-repressor. The HEK-Luc cell line carries a constitutively activeluciferasereportergenedrivenbythe CMV promoter. B. HEK-MYB-Luc and HEK- Luccellsgrownfor12hinpresenceof1μg/ ml doxycyclin and the indicated con- centrations of monensin were analyzed for luciferase activity.    Columns show the mean fluorescence and viability with standard deviations, normalized to cells cultured without monensin. Asterisks indicate sta- tistical significance compared to the un- treated control (**p < 0.01, ***p < 0.001, Student\'s t-test). C. H

 W. Zhou, et al. Cancer Letters 483 (2020) 75–86 elicitingEMTandcellproliferationandmigrationconvergeonAKTand 3.5. MICAL2 nuclear export depended on myosin-9 in LUAD cells myosin-9. Reduced expression of myosin-9 and repressed phosphor- ylation of AKT and GSK-3β in the MICAL2-knockdown PC-9 were To further investigate the factors mediating the nucleocytoplasmic confirmed, indicating the potential role of these signaling pathways in shuttling of MICAL2 cck-8 structure , immunoprecipitation (using anti-MICAL2 anti- mediating MICAL2 function in LUADs (Fig. 2G). body) coupled with mass spectrometry (IP-MS) was applied to search To further verify the tumor promoting role of MICAL2 in LUADs, a for the endogenous MICAL2-binding proteins in PC-9 cells. After sub- lentiviral gene delivery system was used to overexpress MICAL2 in the tracting the non-specific binding proteins using isotype-controlled IP- MICAL2-low A549 cells with undetectable levels of endogenous MS, 108 proteins wer

  r> r> M.V. Yusenko, et al. Cancer Letters 479 (2020) 61–70 Fig. 3. Monensin induces differentiation and apoptosis in myeloid cell lines. NB4 and HL60 cells were cultured for 2 days in the presence of the indicated concentrations of monensin. The cells were then stained with against CD11b (A) or CD14 (B) or with annexinV (C) and analyzed by flow cytometry. Columnsindicatethepercentageofpositivecells.Asterisksindicatestatisticalsignificance(*p < 0.05,**p < 0.01,***p < 0.001,Student\'st-test).PanelDshows May-Grünwald-Giemsa staining of the indicated cell lines cultivated for 48 h without or with 100 nM monensin Cell Counting Kit-8 inhibitor . 3.6. Primary murine AML cells are more sensitive to monensin than normal hematopoietic tumor cells frequently expressing oncogenic MYB-NFIB hematopoietic progenitor cells fusion proteins as a result of chromosomal translocations [13]. We in- itially performed viability assays with short-term cultured tumor cells To comp

 C. Zhao, et al. Cancer Letters 481 (2020) 15–23 Fig.4. Prodigiosinsensitizescolorectalcancercellsto5-Fu-inducedcelldeathpartlythroughacaspase-dependentprocess.(A)ImmunoblottingforLC3and SQSTM1 were used to observe autophagy. SW480 were pretreated with prodigiosin (0.1 μM) for 1 h, followed by 5-Fu (50 μM) for another 12 h LC3B-II and SQSTM1 proteinlevelswereanalyzedusingwesternblotting.GAPDHwasusedastheloadingcontrol.5-Fu,5-fluorouracil. (BandC)Prodigiosin sensitizesHCT116 andSW480cellsto5-Fu-inducedcelldeath.Thecellsweretreatedwith5-Fu(50μM)inthepresenceorabsenceof0.1μMprodigiosinfor48h,andMTSassayswere performedtoassesscellviability.Theresults(mean ± SD)arerepresentativeof3independentexperiments;**,P < 0.01.(D)Apoptosisinductionbycotreatment withprodigiosinand5-Fu.TheHCT116cellswerecotreatedwith0.1μMprodigiosinand50μM5-Fufor48h,andapoptoticcellsweredetectedusingAnnexinV/PI staining followed by fluorescence microscopy imaging (left) and flow cytometry (right). Representat

 D. Zhu, et al. Cancer Letters 477 (2020) 107–121 Fig. 5. Dysbindin,a directtargetofmiR-342–3p, isnegatively regulatedbymiR-342–3pinPDAC.(A)Thetop5predictedcandidate miRNAs(miR-199a-5p,miR- 199b-5p,miR-182–5p,miR-377–3pandmiR-342–3p)targetingdysbindinaccordingtoTargetScansoftware.(B)Predictedpairingtothetargetregioninthe3′UTRof dysbindin(top)andthefivepredictedcandidatemiRNAs(bottom).(C)WesternblotanalysisshowingdysbindinproteinlevelsinAspc-1cellstransfectedwiththe fivecandidatemiRNAmimics(miR-199a-5p,miR-199b-5p,miR-182–5p,miR-377–3pandmiR-342–3p).Thedataarepresentedasthemean ± SEM.(D)AfterPDAC cells(Aspc-1,Capan-2andBxpc-3)weretransfectedwithmiR-342–3pmimicormimiccontrol,Westernblotanalysiswasperformedtodeterminedysbindinprotein levels.Thedataarepresentedasthemean ± SEM.(E)ThreepredictedbindingsitesofmiR-342–3pinthe3′UTRofdysbindinmRNA.MiR-342–3pdecreasedthe luciferaseactivityoftheWTdysbindinreporterbutnotthemutantreporter.Thedataarepresentedasthemean ± SEM.*P < 0.05,**