they were fixed with 4% paraf- was 3.44%

     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 after 488, and 647 (Bioss, Beijing, China) for 1 h. Subsequently, cells were SSL6 treatment for 24 h (Fig. 2E). However, SSL6 treatment for 72 h stained with DAPI for 15 min BAY-598. The image of microscope slides were inhibited CD47 expression in Huh-7 and MHCC97H cells (Fig. 2F). Of taken by a Leica TCS SP5 laser scanning confocal microscope (Leica note, SSL6 also impaired the SFN-enhanced CD47 mRNA expression Microsystems, Wetzlar, Germany). (Fig. 2G). These data demonstrate that SSL6 co-localizes and inhibits CD47 in HCC cells. 2.11. Immunohistochemistry 3.3. CD47deficiencyenhancesHCCcellsensitivitytoSFN The immunohistochemistry of xenograft tumor sections were con- ducted as described previously [32] cck8 inhibitor. The slides were respectively We wondered whether the down-regulated CD47 would result in stained with hematoxylin-eosin (HE) and primary CD47 antibody decreasedproliferationorenhancedapoptosis AngiotensinI.However,knockdownof (ab218810, abcam) and imaged under a light microscope (DMI3000B, CD47 by CRISPR/Cas9 did not affect the proliferation and apoptosis of Leica Microsystems). Huh-7 and MHCC97H cells, which was consistent with the results of SSL6 alone (Fig. 3A–C and Supplementary Fig. 2). Of interest, CD47 2.12. Statisticalanalysis deficiency promoted SFN-induced cell death (Fig. 3D) and decreased IC50 (IC50 values for SFN in Huh-7 sgCD47 cells and NC cells were Data were statistically analyzed by two-tailed unpaired Student\'st- 4.013 and 5.207 μM, respectively; IC50 values for SFN in MHCC97H test and one-way ANOVA using GraphPad Prism8.0 (GraphPad sgCD47 cells and NC cells were 4.209 and 6.537 μM, respectively) Software, Inc., La Jolla, CA, USA) and Excel 2010 (Microsoft). All ex- (Fig. 3E). These results suggest that CD47 reduces SFN sensitivity of periments included at leastthree independent samples. The calculation HCC cells. formula of increase percentage of avs. b was(‾a -‾b) × 100%/‾b. The calculation formula of reduction percentage of a vs. b was (‾b 3.4. SFN-enhancedglycolysisinHCCcellsisimpairedbySSL6 -‾a) × 100%/‾b. Values satisfying * p < 0.05, ** p < 0.01, and *** p < 0.001 were considered statistically significant. Literature have shown that SFN-strengthened glycolysis is a major cause of SFN tolerance [34]. We hypothesized whether SSL6 could 3. Results regulate glycolysis to enhance the SFN sensitivity of HCC cells. As ex- pected,SFNincreasedextracellularacidificationrate(ECAR),aswellas 3.1. SSL6sensitizesHCCcellstoSFN the production of lactate in Huh-7 and MHCC###http://www.glpbio.com/simage/GA11233-L-NAME-hydrochloride-3.png####97H cells, while SSL6 inhibited these indices and reversed SFN-enhancedECAR (Fig.4A)and Recombinant SSL6 was synthesized with cell-free protein synthesis lactate (Huh-7 cells: the increase percentage of SFN vs. control was method, using an Escherichia coli cell extract (Fig. 1A). After the SSL6 49.67%, p = 0.0039; the reduction percentage of SSL6vs. control was protein was purified, SDS-PAGE electrophoresis analysis identified the 13.67%, p = 0.0017; the reduction percentage of SFN + SSL6vs. SFN high purity of SSL6 protein (Fig. 1B). The calculated molecular weight was 20.84%, p = 0.0367. MHCC97H cells: the increase percentage of of the protein was 33KD. The corresponding bands were also detected SFNvs. control was 15.25%, p = 0.0015; the reduction percentage of by western-blot, which validated the successful synthesis and pur- SSL6vs. control was 19.83%, p = 0.0007; the reduction percentage of ification (Fig. 1C). SFN+SSL6vs.SFNwas10.89%,p=0.0023)(Fig.4B).Moreover,the Since SSL6 is a ligand for CD47 [29], we first determined the ex- SFN-upregulatedmRNAlevelsofkey moleculesin glycolysis,including pression of CD47 in 7 HCC cell lines and LO2 hepatocytes. Compared HIF1A, HK2, PFKP and GLUT1 were consistently dampened by SSL6 with LO2 cells, only the Huh-7 and MHCC97H cells expressed sig- treatment (Fig. 4C–F and Supplementary Fig. 3), which was consistent nificantly elevated CD47 in flow cytometry and western-blotting ex- with the protein results (Fig. 4G).

 

 periments (Supplementary Fig. 1A and 1B). Therefore, Huh-7 and The glycolysis was also compared between negative control (NC) MHCC97H cells were selected for the following experiments. The IC50 and CD47 knockdown (sgCD47) groups. Similar to the data from SSL6 valuesforSFNinHuh-7andMHCC97Hcellswere4.718and6.537μM, treatment, CD47 deficiency lead to down-regulated Warburg effect respectively (Supplementary Fig. 1C). (Fig. 4J Huh-7 cells: the reduction percentage of sgCD47 vs. NC was We next evaluated the effects of SSL6 on the proliferation and 28.15%, p = 0.0023. MHCC97H cells: the reduction percentage of apoptosis of HCC cells, and found that the proliferation, cell cycle and sgCD47 vs. NC was 38.03%, p = 0.0009) (Fig. 4H–J). Together these apoptosis of Huh-7 and MHCC97H cells were not significantly altered data suggest that SSL6 enhances the sensitivity of HCC cells to SFN by by SSL6 treatment alone (Supplementary Fig. 1D-F). Of interest, SSL6 blunting glycolysis in HCC cells. dose-dependently promoted SFN-induced death of Huh-7 and MHCC97H cells (Fig. 1D and E). Together these results indicate that 3.5. SSL6suppressesCD47/PI3K/Akt/HIF-1signalingpathwaytodown- SSL6 sensitizes HCC cells to SFN. regulateWarburgeffect 3.2. SSL6inhibitsCD47inHCCcells Glycolysis is regulated by multiple signaling pathways [35–37], 2+ among which, CD47/PI3K/Akt/HIF-1 [38–41], PLCγ-driven Ca ToaddresswhetherCD47wasinvolvedinthemechanismbywhich transient[42],andPKA/AMPK[43,44]aremainpathwaysinvestigated. SSL6 promoted SFN sensitivity of HCC cells, we examined CD47 ex- To explore the underlying mechanism by which SSL6 suppressed pression in Huh-7 cells upon SFN or SSL6 treatment. SFN increased Warburg effect in HCC cells, we evaluated these potential signaling CD47 expression in a dose-dependent manner (Fig. 2A), while SSL6 pathways.WefoundthatCD47/PI3K/Akt/HIF-1,butnotPLCγorPKA/ significantlyinhibitedCD47levelinHuh-7cells(Fig.2B).Weemployed AMPK, was down-regulated by SSL6 (Fig. 5A–C). Consistently, 37