Journal: Journal of Cellular and Molecular Medicine

Article Title: ASPP2 suppresses tumour growth and stemness characteristics in HCC by inhibiting Warburg effect via WNT /β‐catenin/ HK2 axis

doi: 10.1111/jcmm.17687

Figure Lengend Snippet: The associations of ASPP2 and HK2 expression with clinicopathologic characteristics in 80 patients with HCC

Article Snippet: We designed three pairs of cDNA oligonucleotides targeting ASPP2 mRNA expression, using web‐based software from Invitrogen and InvivoGen Inc. After synthesis, we inserted these double‐strand oligos into the vector pENTR/U6 (Invitrogen) and sequenced the resulting plasmids to ensure the shRNA construct targeted human ASPP2 expression or were scrambled, which were generated and designated as LV‐shAspp2 and LV‐shNon.

Techniques: Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: ASPP2 suppresses tumour growth and stemness characteristics in HCC by inhibiting Warburg effect via WNT /β‐catenin/ HK2 axis

doi: 10.1111/jcmm.17687

Figure Lengend Snippet: Downregulation of ASPP2 enhance tumour aerobic glycolysis in HCC cells. (A) Non‐targeted metabolomics were measured by GC–MS methods, using the internal standard strategy as described in methods. Correlation matrix analysis show changes in metabolomics between HCC‐LM3 infected with LV‐shAspp2 and LV‐shNon. Arrows illustrate pyruvic acid, lactate acid and glucose were significant correlation in HCC‐LM3 infected with LV‐shAspp2 and LV‐shNon. (B) Pyruvic acid production, lactate production and glucose consumption in HCC‐LM3 and Hep‐G2 infected with LV‐shAspp2 or LV‐shNon and Huh‐7 cells transfected with ASPP2 plasmid or control. (C) Effects of ASPP2 on oxygen consumption ratio (OCR) and extracellular acid ratio (ECAR) in HCC‐LM3. O, oligomycin; F, FCCP (carbonyl cyanide 4‐[trifluoromethoxy] phenylhydrazone); A&R, antimycin A and rotenone; Glu, glucose; O, oligomycin; 2‐DG, 2‐deoxyglucose. All data are shown as the mean ± SEM of 3 independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: We designed three pairs of cDNA oligonucleotides targeting ASPP2 mRNA expression, using web‐based software from Invitrogen and InvivoGen Inc. After synthesis, we inserted these double‐strand oligos into the vector pENTR/U6 (Invitrogen) and sequenced the resulting plasmids to ensure the shRNA construct targeted human ASPP2 expression or were scrambled, which were generated and designated as LV‐shAspp2 and LV‐shNon.

Techniques: Gas Chromatography-Mass Spectrometry, Infection, Transfection, Plasmid Preparation

Journal: Journal of Cellular and Molecular Medicine

Article Title: ASPP2 suppresses tumour growth and stemness characteristics in HCC by inhibiting Warburg effect via WNT /β‐catenin/ HK2 axis

doi: 10.1111/jcmm.17687

Figure Lengend Snippet: ASPP2 Decreases the expression of glycolytic enzymes in HCC cells. (A) The mRNA levels of HK2, PFKFB3 and PKM2 were determined after ASPP2 knockdown in HCC‐LM3, Hep‐G2 and Hep‐3B cells. The mRNA levels of HK2, PFKFB3 and PKM2 were determined after ASPP2 overexpression in Huh‐7 cell. (B) The protein levels of HK2 and PKM2 were determined after ASPP2 knockdown in HCC‐LM3, Hep‐G2 and Hep‐3B cells, as well as in Huh‐7 cells with overexpressed‐ASPP2. The protein expression had been quantified and presented in a bar graph. The same bands of β‐Actin in Hep‐3B cells are used in (B) and Figure C. And, the same bands of β‐Actin in Huh‐7 cells are used in (B) and Figure D. All data are shown as the mean ± SEM of 3 independent experiments. * p < 0.05; ** p < 0.01

Article Snippet: We designed three pairs of cDNA oligonucleotides targeting ASPP2 mRNA expression, using web‐based software from Invitrogen and InvivoGen Inc. After synthesis, we inserted these double‐strand oligos into the vector pENTR/U6 (Invitrogen) and sequenced the resulting plasmids to ensure the shRNA construct targeted human ASPP2 expression or were scrambled, which were generated and designated as LV‐shAspp2 and LV‐shNon.

Techniques: Expressing, Over Expression

Journal: Journal of Cellular and Molecular Medicine

Article Title: ASPP2 suppresses tumour growth and stemness characteristics in HCC by inhibiting Warburg effect via WNT /β‐catenin/ HK2 axis

doi: 10.1111/jcmm.17687

Figure Lengend Snippet: ASPP2 depresses β‐catenin and its downstream target expression. After treatment with XAV‐939 (10 μM) or ICG‐001 (10 μM) for 24 hours, the protein level of β‐catenin and its downstream target were analysed using Western blot in (A) HCC‐LM3 cells and (B) Hep‐G2 cells with ASPP2 knockdown, as well as in (C) Huh‐7 cells with ASPP2 overexpression, compared to relative control cells. The subcellular distribution of β‐catenin in cytoplasm (marked as C) and nucleus (marked as N) of HCC‐LM3 cells (D) and Hep‐G2 cells (E) after ASPP2 knockdown, as well as (F) Huh‐7 cells with ASPP2 overexpression. α‐tubulin and Lamin B were served as internal control of cell cytoplasm and nucleus accordingly. (G) Schematic representation for the mechanism of ASPP2 mediated glycolysis in HCC. Schematic illustrates how ASPP2 inhibit β‐catenin and its downstream target expression, c‐myc, CyclinD1 and P21, as well as glycolytic key enzymes HK2 and PKM2, which reduce the production of lactate acid and pyruvic acid in glycolysis

Article Snippet: We designed three pairs of cDNA oligonucleotides targeting ASPP2 mRNA expression, using web‐based software from Invitrogen and InvivoGen Inc. After synthesis, we inserted these double‐strand oligos into the vector pENTR/U6 (Invitrogen) and sequenced the resulting plasmids to ensure the shRNA construct targeted human ASPP2 expression or were scrambled, which were generated and designated as LV‐shAspp2 and LV‐shNon.

Techniques: Expressing, Western Blot, Over Expression

Journal: Journal of Cellular and Molecular Medicine

Article Title: ASPP2 suppresses tumour growth and stemness characteristics in HCC by inhibiting Warburg effect via WNT /β‐catenin/ HK2 axis

doi: 10.1111/jcmm.17687

Figure Lengend Snippet: ASPP2 reduces the Warburg effect by inhibition of β‐catenin signalling. (A–C) Glucose, lactate and pyruvic acid (PK) production in HCC‐LM3, Hep‐G2 cells infected with LV‐shAspp2 or LV‐shNon and Huh‐7 cells with ASPP2 overexpression, after treatment with XAV‐939 (10 μM) or ICG‐001 (10 μM) for 24 h. (D) HCC‐LM3 cells infected with LV‐shAspp2 or LV‐shNon, after treatment with XAV‐939 (10 μM) or ICG‐001 (10 μM) for 24 h. Effects of ASPP2 on oxygen consumption ratio (OCR) and extracellular acid ratio (ECAR). All data are shown as the mean ± SEM of 3 independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: We designed three pairs of cDNA oligonucleotides targeting ASPP2 mRNA expression, using web‐based software from Invitrogen and InvivoGen Inc. After synthesis, we inserted these double‐strand oligos into the vector pENTR/U6 (Invitrogen) and sequenced the resulting plasmids to ensure the shRNA construct targeted human ASPP2 expression or were scrambled, which were generated and designated as LV‐shAspp2 and LV‐shNon.

Techniques: Inhibition, Infection, Over Expression

Journal: Journal of Cellular and Molecular Medicine

Article Title: ASPP2 suppresses tumour growth and stemness characteristics in HCC by inhibiting Warburg effect via WNT /β‐catenin/ HK2 axis

doi: 10.1111/jcmm.17687

Figure Lengend Snippet: Warburg effect is essential for maintaining tumour‐initiating capability in ASPP2‐depleted HCC cells. (A) Left: Representative images of the first spheroid formation in HCC‐LM3 cells with 3‐bp (60 μM) or 2‐DG (0.1 μM) after knocking‐down of ASPP2. Right: the number of spheres formation per 2000 cells. Scale bar, 100 μm. (B) Left: Representative images of the tertiary spheres formation in HCC‐LM3 and Hep‐G2 cells with 2‐DG (0.1 μM) after knocking‐down of ASPP2. Right: the number of spheres formation per 2000 cells. Scale bar, 100 μm. (D) qRT‐PCR gene expression of Epcam, Oct‐4, Abcg2 and CD44 in HCC‐LM3 and HepG2 cells transfected with the indicated lentivirus and treated with 2‐DG (0.1 μM). (D) Cell viability of infected HCC‐LM3 and Hep‐G2 cell treated with 5‐FU (10 ug/mL) and Oxaliplatin (1 μM) with or without 2‐DG (0.1 μM) was analysed by CCK‐8 assay in 24, 48 h

Article Snippet: We designed three pairs of cDNA oligonucleotides targeting ASPP2 mRNA expression, using web‐based software from Invitrogen and InvivoGen Inc. After synthesis, we inserted these double‐strand oligos into the vector pENTR/U6 (Invitrogen) and sequenced the resulting plasmids to ensure the shRNA construct targeted human ASPP2 expression or were scrambled, which were generated and designated as LV‐shAspp2 and LV‐shNon.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Infection, CCK-8 Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: ASPP2 suppresses tumour growth and stemness characteristics in HCC by inhibiting Warburg effect via WNT /β‐catenin/ HK2 axis

doi: 10.1111/jcmm.17687

Figure Lengend Snippet: Downregulation of ASPP2 promoted tumour growth in vivo by activating Warburg effect. (A) Representative dissected tumours from nude mice treated with physiological saline or with 2‐DG (500 mg/kg) for 3 weeks (left) and corresponding volume measurement (right), asterisk (*) indicates p < 0.05. (B) Whole body bioluminescent images of representative HCC‐LM3‐luc tumour xenograft mice were determined by the Xenogen IVIS 100 imaging System. (C) Representative micro‐PET/CT images of xenograft tumours induced in nude mice by LV‐shAspp2 or LV‐shNon HCC‐LM3 cells, with the arrow representing the tumour site. (D) HK2 and PKM2 were detected by immunohistochemical staining in tumour xenograft mice tissue. Representative pictures are shown (Scale bars: 100 μm). (E) The mRNA of HK2 and PKM2 were examined by q‐PCR in mice tumour tissues in different treated group randomly, values are given as the means ± SD of 3 mice

Article Snippet: We designed three pairs of cDNA oligonucleotides targeting ASPP2 mRNA expression, using web‐based software from Invitrogen and InvivoGen Inc. After synthesis, we inserted these double‐strand oligos into the vector pENTR/U6 (Invitrogen) and sequenced the resulting plasmids to ensure the shRNA construct targeted human ASPP2 expression or were scrambled, which were generated and designated as LV‐shAspp2 and LV‐shNon.

Techniques: In Vivo, Imaging, Micro-PET, Immunohistochemical staining, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: ASPP2 suppresses tumour growth and stemness characteristics in HCC by inhibiting Warburg effect via WNT /β‐catenin/ HK2 axis

doi: 10.1111/jcmm.17687

Figure Lengend Snippet: Expression of ASPP2 correlates negatively with HK2 in surgical specimens of HCC. (A) ASPP2 and HK2 were detected by immunohistochemical staining in 80 HCC samples. Representative pictures are shown for four patients. (Scale bars: 100 μm). (B) Table of ASPP2 and HK2 high and low expression according to immunohistochemistry scoring. Significant negative correlation between ASPP2 and HK2 expression was found, Fisher's test P < 0.05. (C) Top: Overall survival (OS) and Bottom: recurrence‐free survival (RFS) rate curves of the analysed four subgroups (ASPP2 low/HK2 low; ASPP2 low/HK2 high; ASPP2 high /HK2 low; ASPP2 high/HK2 high), ( n = 80). Kaplan–Meier analysis showed RFS ( p < 0.05) and OS ( p < 0.05) were significantly best amongst patients with ASPP2‐high and HK2‐low expression

Article Snippet: We designed three pairs of cDNA oligonucleotides targeting ASPP2 mRNA expression, using web‐based software from Invitrogen and InvivoGen Inc. After synthesis, we inserted these double‐strand oligos into the vector pENTR/U6 (Invitrogen) and sequenced the resulting plasmids to ensure the shRNA construct targeted human ASPP2 expression or were scrambled, which were generated and designated as LV‐shAspp2 and LV‐shNon.

Techniques: Expressing, Immunohistochemical staining, Staining, Immunohistochemistry

Journal: Journal of Cellular and Molecular Medicine

Article Title: ASPP2 suppresses tumour growth and stemness characteristics in HCC by inhibiting Warburg effect via WNT /β‐catenin/ HK2 axis

doi: 10.1111/jcmm.17687

Figure Lengend Snippet: Univariate analyses of factors associated with recurrence‐free Survival (RFS) and overall survival (OS)

Article Snippet: We designed three pairs of cDNA oligonucleotides targeting ASPP2 mRNA expression, using web‐based software from Invitrogen and InvivoGen Inc. After synthesis, we inserted these double‐strand oligos into the vector pENTR/U6 (Invitrogen) and sequenced the resulting plasmids to ensure the shRNA construct targeted human ASPP2 expression or were scrambled, which were generated and designated as LV‐shAspp2 and LV‐shNon.

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: ASPP2 suppresses tumour growth and stemness characteristics in HCC by inhibiting Warburg effect via WNT /β‐catenin/ HK2 axis

doi: 10.1111/jcmm.17687

Figure Lengend Snippet: Multivariate analyses of factors associated with recurrence‐free Survival (RFS) and overall survival (OS)

Article Snippet: We designed three pairs of cDNA oligonucleotides targeting ASPP2 mRNA expression, using web‐based software from Invitrogen and InvivoGen Inc. After synthesis, we inserted these double‐strand oligos into the vector pENTR/U6 (Invitrogen) and sequenced the resulting plasmids to ensure the shRNA construct targeted human ASPP2 expression or were scrambled, which were generated and designated as LV‐shAspp2 and LV‐shNon.

Techniques: