- 14-0138-82 - Invitrogen Antibodies ANPEP antibody

Submitted references Targeting glutaminase 1 attenuates stemness properties in hepatocellular carcinoma by increasing reactive oxygen species and suppressing Wnt/beta-catenin pathway.

Li B, Cao Y, Meng G, Qian L, Xu T, Yan C, Luo O, Wang S, Wei J, Ding Y, Yu D
EBioMedicine 2019 Jan;39:239-254

LncBRM initiates YAP1 signalling activation to drive self-renewal of liver cancer stem cells.

Zhu P, Wang Y, Wu J, Huang G, Liu B, Ye B, Du Y, Gao G, Tian Y, He L, Fan Z
Nature communications 2016 Dec 1;7:13608

Model of fibrolamellar hepatocellular carcinomas reveals striking enrichment in cancer stem cells.

Oikawa T, Wauthier E, Dinh TA, Selitsky SR, Reyna-Neyra A, Carpino G, Levine R, Cardinale V, Klimstra D, Gaudio E, Alvaro D, Carrasco N, Sethupathy P, Reid LM
Nature communications 2015 Oct 6;6:8070

ZIC2-dependent OCT4 activation drives self-renewal of human liver cancer stem cells.

Zhu P, Wang Y, He L, Huang G, Du Y, Zhang G, Yan X, Xia P, Ye B, Wang S, Hao L, Wu J, Fan Z
The Journal of clinical investigation 2015 Oct 1;125(10):3795-808

Induction of aminopeptidase N/CD13 on human lymphocytes after adhesion to fibroblast-like synoviocytes, endothelial cells, epithelial cells, and monocytes/macrophages.

Riemann D, Kehlen A, Thiele K, Löhn M, Langner J
Journal of immunology (Baltimore, Md. : 1950) 1997 Apr 1;158(7):3425-32

Human myeloid plasma membrane glycoprotein CD13 (gp150) is identical to aminopeptidase N.

Look AT, Ashmun RA, Shapiro LH, Peiper SC
The Journal of clinical investigation 1989 Apr;83(4):1299-307

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Supportive validation

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Experimental details: Staining of normal human peripheral blood cells with 0.5 µg of Mouse IgG1 Isotype Control Purified (Product # 14-4714-82) (open histogram) or 0.5 µg of Anti-Human CD13 Purified (filled histogram) followed by Anti-Mouse IgG FITC (Product # 11-4011-85). Cells in the monocyte (left) and granulocyte (right) gates were used for analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 LncBRM is highly expressed in HCC tumours and liver CSCs. ( a ) The indicated lncRNAs were silenced using pSiCoR lentivirus, followed by sphere formation assays. *, **, for Hep3B cells, Hep3B shlncBRM versus Hep3B shCtrl; #, ##, for Huh7 cells, Huh7 shlncBRM versus Huh7 shCtrl. ( b ) Total RNAs were extracted from peri-tumour (P) and tumour (T) tissues, followed by northern blotting. ACTB served as a loading control. ( c ) Primary HCC samples were prepared for examination of lncBRM expression using RT-qPCR. aHCC, advanced HCC; eHCC, early HCC. ( d ) LncBRM was detected by in situ hybridization. LncBRM highly expressed cells (middle panel) and lncBRM photon intensity (right panel) were calculated by Image-Pro Plus 6 and shown as scatter plot (means+-s.e.m.). Scale bars, 100 mum. ( e ) Liver CSCs (CD13 + CD133 + ) and non-CSCs (CD13 - CD133 - ) were sorted from HCC samples, followed by detection of lncBRM using RT-qPCR (left panel). Oncospheres and non-spheres derived from HCC primary tumour cells were analysed similarly. Expression levels of lncBRM were normalized to that of non-tumour sample 17 as a baseline level. ( f ) lncBRM was examined in oncospheres and non-spheres with northern blotting. N, non-sphere; S, sphere. ( g ) Non-spheres and spheres were stained with lncBRM probes and CD13 antibody for confocal microscopy. Scale bars, 20 mum. ( h ) Nucleocytoplasmic fractionation of oncosphere cells was performed and followed by immunoblotting (upper panel) and RT-qP

Supportive validation

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Experimental details: Figure 2 Characterization of a transplantable hFL-HCC tumour line. ( a ) Representative flow cytometric findings of hFL-HCC cells, gated-mouse-H2K d -negative, were done. Antigens expressed by a significant percentage of the cells included CD44 (61.4%); CD49f (25.4%); CD24 (32.9%); CD13 (12.5%); c-KIT (12.0%); E-cadherin (12.0%); and OSMR (10.7%). Other antigens found routinely but in a smaller percentage of cells included CXCR4 (4.8%); EpCAM (4.3%); CD133 (2.3%); TROP-2 (1.4%); and ICAM1 (0.5%). ( b ) Double staining of LGR5 and EpCAM in hFL-HCC cells. LGR5+ cells accounted for 68.9% of the cells in the tumours. Of these, only 1.1% were also EpCAM+. ( c ) Immunofluorescence assay on hFL-HCC xenotransplantable tumour demonstrated strong expression of LGR5 and an absence of EpCAM. ( d ) IHC assays on the xenotransplantable tumour line. The survey included assays for endodermal stem/progenitor transcription factors and markers (SOX17, SOX9, CD44, LGR5 and CK19); pluripotency genes (NANOG, OCT4, KLF4, SOX2 and BMI1); hepatic and other markers (HepPar-1, CK18, CK7, SHH and CD68); and pancreatic/endocrine markers (PDX1, NGN3 and NIS), which were strongly expressed. EpCAM was essentially negative, and alpha-fetoprotein was negative. The scale bar, 25 mum ( c , d ).

Supportive validation

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Experimental details: Fig. 5 Targeting GLS1 suppresses cancer stem cell properties. (a) Effect of glutamine on the expression of stemness genes. HCCLM3 and SMMC-7721 were cultured in DMEM with the absence of glutamine for 24, 48, or 72 h. Cell lysates were subjected to Western blot to measure the expression of stemness markers. (b) HCCLM3 cells were treated with 5, 10, or 20 muM 968 or BPTES for 48 h, then Western blot was employed to detect the expression of stemness markers. (c-d) Glutamine deprivation or 968 treatment for 48 h resulted in a decrease of CD13 expression as evaluated by flow cytometry. (e) Colony formation assay was performed to assess the effect of glutamine withdrawal and GLS1 inhibitors (968 or BPTES) on tumorigenicity. (f) Colony formation assay and quantitative analysis were performed in HCCLM3 and SMMC-7721 cells with GLS1 knockout. (g-h) 968 treatment reduced the number and size of the tumorspheres in Hep3B as measured by sphere-formation assays. Scale bars: 200 mum (i) The expression changes of stemness markers in cell lines stably overexpressing empty vector (lvCON), KGA (lvKGA) or GAC (lvGAC) isoform by lentiviral. (j-k) Colony formation assay and quantitative analysis were performed in lvCON, lvKGA, and lvGAC cell lines. Data are presented as mean +- SD. **, p < .01; ***, p < .001. Fig. 5

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