SSL6promotesSFN-inducedapoptosis ofHCCcells.(A)Theprotocolfor SSL6synthesis.(B)Western-blot

 
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, cell suspension was prepared with Annexin-V 4 Binding Buffer and stained with FITC Annexin-V Apoptosis Detection 2 × 10 cell/mL suspension was inoculated into a 96-well plate Kit (640922, Biolegend) in the dark for 15 min at room temperature. (100 μL every well) overnight at 37 °C. Cell supernatant was collected After starvation for 12 h to synchronize cell cycle, PI/RNase Staining for evaluating the lactate concentration according to manufacturer\'s Buffer (550825, BD Biosciences, San Diego, US) was used for cell cycle instructions (L256, Dojindo Laboratories). evaluation. After staining, the CD47 expression and cell death were examined by FACSCanto II system (BD Biosciences). The mean fluor- 2.9. ECARmeasurements escenceintensity(MFI)wascalculatedwithFlowJosoftware(TreeStar) and ModiFit LT v4 Salvinorin A.0 software (Verify Software House, Topsham, ME, Cells were seeded into a Seahorse XFp Cell Culture Mini Microplate 34
X.Zhang,etal. Cancer Letters 481 (2020) 32–44 Fig. 2. SSL6 inhibits CD47 expression in HCC cells. (A,B) Flow cytometry of CD47 on the cell surface of Huh-7 upon different doses of SSL6 (A) and SFN (B) treatment. (C,D) Immunofluorescence of SSL6 and CD47 localization after MHCC97H were treated with SSL6 (50 μg/mL) for 24 h(C) or 72 h(D). (E,F) CD47 protein expression in Huh-7 cells after treated with PBS or SSL6 (10 μg/mL) for 24 h (E) or 72 h (F). (G)CD47 mRNA expression in Huh-7 (Left) and MHCC97H (Right) cells after treated with SFN (5 μM) or SSL6 (10 μg/mL) for 72 h. Data are expressed as means ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. 3 (Agilent, CA, USA) at 5 × 10 cells/well. After were washed by Test Kit (103020-100, Agilent) was used according to manufacturer Seahorse XF Base Medium (102334-100, Agilent) supplemented with instructions. Next, ECAR was measured with Seahorse XFp Instrument 2 mM glutamine (103579, Agilent), the Seahorse XFp Glycolysis Stress (Agilent). 35
X.Zhang,etal. Cancer Letters 481 (2020) 32–44 Fig.3. KnockdownofCD47promotesHCCcellsensitivitytoSFN Z-VAD-FMK.(A-C)CRISPR-Cas9againstCD47wasperformedwithHuh-7andMHCC97Hcells,qPCR(A),flow cytometry(B)andwestern-blot(C)wereemployedtodeterminetheexpressionofCD47.NCdenotesnegativecontrol,sgCD47denotessgRNAtargetinghumanCD47. (D)FlowcytometrydetectionofcelldeathafterNCandsgCD47Huh-7cellsweretreatedwithvariousdosesofSFNfor72h.(E)IC50ofHCCcellsinNCandsgCD47 groups. Data are expressed as means ± SEM (n = 3). **p < 0.01, ***p < 0.001. 36
r> X.Zhang,etal. Cancer Letters 481 (2020) 32–44 Fig. 4. SSL6 dampens SFN-enhanced glycolysis in HCCcells through blocking CD47. (A) After HCCcells were treated with SFN (5 μM) and/orSSL6 (10μg/mL) for 72h CCK8,theextracellularacidificationrate(ECAR)weremeasuredusingaSeahorseXFpanalyzer.(B)AftertreatmentofHuh-7(Left)andMHCC97H(Right)cellswith SFN(5μM)and/orSSL6(10μg/mL)for72h,theintracellularlacticacidlevelwereexaminedwithLactateAssayKit.(C–F)AftertreatmentofHuh-7cellswithSFN (5μM)and/orSSL6(10μg/mL)for72h,themRNAlevelsofHIF1A,HK2,PFKP,andGLUT1wereanalyzed.(G)AftertreatmentofHuh-7cellswithSFN(5μM)and/ orSSL6(10μg/mL)for72h,theproteinexpressionofCD47,HIF-1α,andkeyenzymesinglycolysiswereassessed.(H)Heatmapofgeneexpressionofkeyenzymesin NCandsgCD47Huh-7cells.(I)Theextracellularacidificationrate(ECAR)ofNCandsgCD47MHCC97Hcells.(J)IntracellularlactatelevelsofNCandsgCD47HCC cells. Data are expressed as means ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. knockdown of CD47 impaired the activation of CD47/PI3K/Akt/HIF-1 (Fig. 5F). LY294002 significantly suppressed SFN-enhanced CD47 and in both Huh-7 and MHCC97H cells (Fig. 5D and E). Moreover, inhibi- glycolysis. LY294002 also blocked the effect of SSL6 on glycolysis and tion of PI3K with LY294002 or blockade of glycolysis with 2-deoxy-D- CD47 expression (Fig. 5G and H). Of interest, SFN down-regulated glucose(2DG)alsoimpairedSSL6effectonsensitizingHCCcellstoSFN CD47uponPI3Kinhibition(Fig.5H),suggestingthatPI3Kwasrequired 38