Journal: Cancers

Article Title: BNIP3L-Dependent Mitophagy Promotes HBx-Induced Cancer Stemness of Hepatocellular Carcinoma Cells via Glycolysis Metabolism Reprogramming

doi: 10.3390/cancers12030655

Figure Lengend Snippet: HBV x protein (HBx) promoted the hepatocellular carcinoma (HCC) xenograft tumors growth via the upregulation of glycolytic metabolism in vivo. The HCC xenograft tumors with or without HBx-expressing were formed by Huh7-/MHCC-97H-pcDNA3.1-HA-HBx or Huh7-/MHCC-97H-pcDNA3.1-HA cells in BALB/c nude mice for 24 days. n = 6. ( A ) The growth curves of tumors were measured every two days. ( B ) Excised tumors were photographed after mice were sacrificed. ( C ) Tumor weights in the four groups. ( D ) The expression level of the cancer stemness related-proteins in the Huh7 xenograft tumors with or without HBx-expressing. The gray value of band was assessed by image-pro plus 6.0. The relative expression level was showed. ( E ) The mRNA levels of the cancer stemness-related genes in the Huh7 xenograft tumors with or without HBx-expressing. ( F ) Immunohistochemical staining of the cancer stemness-related proteins in the Huh7 xenograft tumors with or without HBx-expressing. Scale bar represents 50 μm. The levels of ATP content ( G ) and lactic acid ( H ) were detected in Huh7 and MHCC-97H xenograft tumors. ( I ) The mRNA levels of glycolysis-related genes in Huh7 xenograft tumors with or without HBx-expressing. The target gene transcription was normalized to ACTB . * P < 0.05 as compared with pc3.1-HA group. pc3.1-HA: pcDNA3.1-HA transfection without HBx-expressing. pc3.1-HA-HBx: pcDNA3.1-HA-HBx transfection with HBx-expressing.

Article Snippet: Human hepatocellular carcinoma Huh7 and MHCC-97H cell lines were obtaineded from ATCC and maintained in our laboratory.

Techniques: In Vivo, Expressing, Immunohistochemical staining, Staining, Transfection

Journal: Cancers

Article Title: BNIP3L-Dependent Mitophagy Promotes HBx-Induced Cancer Stemness of Hepatocellular Carcinoma Cells via Glycolysis Metabolism Reprogramming

doi: 10.3390/cancers12030655

Figure Lengend Snippet: HBx promoted cancer stemness phenotype of the HCC cells. ( A – E ) The two HCC cell lines without or with HBx-expressing were transiently transfected with pcDNA3.1 or pcDNA3.1-HBx (0.5, 1 μg/mL) for 8 h and then restored culture for another 48 h. ( A , B ) The expression levels of cancer stemness-related proteins ( A ), and the mRNA levels of cancer stemness-related genes ( B ) in two HCC cells without or with HBx-expressing. The target gene transcription was normalized to ACTB . ( C ) Percentage of the sorted SP (R1 gate) in two HCC cells without or with HBx-expressing (Left). Quantitative results were shown in bar graph (Right). SP: side population. R1 gate represented SP cells. ( D , E ) The self-renewal capacity was analyzed by anchorage-independent growth assay ( D ) and colony formation assay ( E ) in two HCC cells without or with HBx-expressing. Magnification (200×). ( F ) The expression levels of cancer stemness-related proteins in two HCC cells transiently transfected with pGEM, pGEM-HBV, and pGEM-HBV X null plasmids. The gray value of band was assessed by image-pro plus 6.0. The relative expression level was shown. * P < 0.05 as compared with Huh7-pc3.1 group. # P < 0.05 as compared with MHCC-97H-pc3.1 group. pc3.1: pcDNA3.1 transfection without HBx-expressing. pc3.1-HBx: pcDNA3.1-HBx transfection with HBx-expressing.

Article Snippet: Human hepatocellular carcinoma Huh7 and MHCC-97H cell lines were obtaineded from ATCC and maintained in our laboratory.

Techniques: Expressing, Transfection, Growth Assay, Colony Assay

Journal: Cancers

Article Title: BNIP3L-Dependent Mitophagy Promotes HBx-Induced Cancer Stemness of Hepatocellular Carcinoma Cells via Glycolysis Metabolism Reprogramming

doi: 10.3390/cancers12030655

Figure Lengend Snippet: Glycolytic metabolism was reprogrammed in HBx-expressing Huh7 cells and liver cancer stem cells (LCSCs). ( A – E ) LCSCs were enriched in Huh7 cells by sphere-formation assay, and normally cultured Huh7 cells served as the parental cells (PT) control group. ( A ) Glucose transport activity was evaluated by Flows cytometry (FCM) (Left). The mean data was shown in bar graph (Right). The levels of intracellular ATP content ( B ), the extracellular lactic acid secretion ( C ), the mRNA levels of glycolysis-related genes ( D ), and the mRNA levels of OXPHOS-related genes ( E ) were detected in PT and LCSCs of Huh7 cells. ( F – J ) Huh7 cells without or with HBx-expressing were transiently transfected with pcDNA3.1 or pcDNA3.1-HBx (1 μg/mL) for 8 h, and followed by restored culture for another 48 h. ( F ) Glucose transport activity was evaluated by FCM (Left). The mean data was shown in bar graph (Right). The levels of the intracellular ATP content ( G ), the extracellular lactic acid secretion ( H ), the mRNA levels of glycolysis-related genes ( I ), and the mRNA levels of OXPHOS-related genes ( J ) were detected in Huh7 cells without or with HBx-expressing. The target gene transcription was normalized to ACTB . * P < 0.05 as compared with PT cells group. # P < 0.05 as compared with pc3.1 group. pc3.1: pcDNA3.1 transfection without HBx-expressing. pc3.1-HBx: pcDNA3.1-HBx transfection with HBx-expressing.

Article Snippet: Human hepatocellular carcinoma Huh7 and MHCC-97H cell lines were obtaineded from ATCC and maintained in our laboratory.

Techniques: Expressing, Tube Formation Assay, Cell Culture, Control, Activity Assay, Cytometry, Transfection

Journal: Cancers

Article Title: BNIP3L-Dependent Mitophagy Promotes HBx-Induced Cancer Stemness of Hepatocellular Carcinoma Cells via Glycolysis Metabolism Reprogramming

doi: 10.3390/cancers12030655

Figure Lengend Snippet: BNIP3L-dependent mitophagy was induced in HBx-expressing HCC cells and LCSCs. ( A – C ) LCSCs were enriched in Huh7 cells by sphere-formation assay, and normally cultured Huh7 cells served as the parental cells (PT) control group. ( A ) Mitochondrial ultrastructures were analyzed by TEM. ( B ) Representative images of the immunofluorescence co-staining for MitoTracker (red), BNIP3L (blue), and LC3B (green). Scale bar represents 10 μm. ( C ) The expression levels of BNIP3L-dependent mitophagy-related proteins. ( D – H ) Huh7 and MHCC-97H cells without or with HBx-expressing were transiently transfected with pcDNA3.1 or pcDNA3.1-HBx (1 μg/mL). ( D ) Representative fluorescent images of Huh7 and HBx-expressing Huh7 cells were transiently transfected with mTagRFP-mWasabi-LC3 with the pretreatment of chloroquine (CQ, 20 μg/mL) or not. ( E ) Mitochondrial ultrastructures in Huh7 and HBx-expressing Huh7 cells were analyzed by TEM. Scale bar represents 2 μm (Left) or 1 μm (Right). ( F ) The protein expression of BNIP3L-dependent mitophagy in HCC cells and their HBx-expressing cells. ( G ) Representative images of the immunofluorescence co-staining for MitoTracker (red), BNIP3L (blue), and LC3B (green) in HCC cells with or without HBx-expressing. The profiles of representative lines trace the intensities of fluorescence signals. Fluorescence curves with line intensity profile generated by Zen 2012 software were shown. ( H ) The protein expression of BNIP3L-dependent mitophagy in cytoplasmic (Cyto) and mitochondrial (Mito) fractions of Huh7 cells and its HBx-expressing cells. The gray value of band was assessed by image-pro plus 6.0. The relative expression level was shown. pc3.1: pcDNA3.1 transfection without HBx-expressing. pc3.1-HBx: pcDNA3.1-HBx transfection with HBx-expressing.

Article Snippet: Human hepatocellular carcinoma Huh7 and MHCC-97H cell lines were obtaineded from ATCC and maintained in our laboratory.

Techniques: Expressing, Tube Formation Assay, Cell Culture, Control, Immunofluorescence, Staining, Transfection, Fluorescence, Generated, Software

Journal: Cancers

Article Title: BNIP3L-Dependent Mitophagy Promotes HBx-Induced Cancer Stemness of Hepatocellular Carcinoma Cells via Glycolysis Metabolism Reprogramming

doi: 10.3390/cancers12030655

Figure Lengend Snippet: Relationship between the BNIP3L-dependent mitophagy and glycolysis metabolism reprogramming in HBx-expressing HCC cells. ( A – D ) Huh7 cells without or with HBx-expressing were transiently transfected with pcDNA3.1 or pcDNA3.1-HBx (1 μg/mL) for 8 h, and pretreated with CCCP (20 μM) for 3 h or not. ( A ) Glucose transport activity was evaluated by FCM. The levels of the intracellular ATP content ( B ), the extracellular lactic acid secretion ( C ), and the mRNA levels of glycolysis-related genes ( D ) were detected. ( E – H ) MHCC-97H cells without or with HBx-expressing were simultaneously transfected with si BNIP3L or siNC (50 nmol/L) for 8 h, and followed by restored culture for another 24 h. ( E ) Glucose transport activity was evaluated by FCM. The levels of the intracellular ATP content ( F ), the extracellular lactic acid secretion ( G ), and the mRNA levels of glycolysis-related genes ( H ) were detected. The target gene transcription was normalized to ACTB. * P < 0.05 as compared with pc3.1+Vehicle group, # P < 0.05 as compared with pc3.1+siNC group, & P < 0.05 as compared with pc3.1-HBx+siNC group. pc3.1: pcDNA3.1 transfection without HBx-expressing. pc3.1-HBx: pcDNA3.1-HBx transfection with HBx-expressing.

Article Snippet: Human hepatocellular carcinoma Huh7 and MHCC-97H cell lines were obtaineded from ATCC and maintained in our laboratory.

Techniques: Expressing, Transfection, Activity Assay

Journal: Cancers

Article Title: BNIP3L-Dependent Mitophagy Promotes HBx-Induced Cancer Stemness of Hepatocellular Carcinoma Cells via Glycolysis Metabolism Reprogramming

doi: 10.3390/cancers12030655

Figure Lengend Snippet: Anti-HBx targeting intervention to intracellular HBx inhibited the hepatocarcinogenesis associated with BNIP3L-dependent mitophagy. ( A – D ) The relative mRNA levels of indicated genes were obtained from NCBI, GEO database (GSE83148). The clinical cohort samples were derived from HBV-infected liver tissues ( n = 122) and normal liver tissues ( n = 6). The heat-map of the relative mRNA levels of cancer stemness-related genes ( A ) and glycolysis-related metabolism genes ( B ), the relative mRNA levels of MAP1LC3B gene ( C ), and the linear correlation of MAP1LC3B with glycolysis-related metabolism genes ( D ) were shown. ( E – J ) Huh7 cells without or with HBx-expressing were transiently transfected with pcDNA3.1 or pcDNA3.1-HBx (1 μg/mL), and then transiently transfected with pTT5 or pTT5-9D11 plasmids (200 ng/mL) for 8 h, and cultured for another 24 h. pTT5-9D11 plasmids encoded anti-HBx, a monoclonal antibody (mcAb), directed against intracellular HBx. ( E ) The expression levels of cancer stemness-related proteins. ( F ) The expression levels of BNIP3L-dependent mitophagy-related proteins. The gray value of band was assessed by image-pro plus 6.0. The relative expression level was shown. ( G ) Glucose transport activity was evaluated by FCM. The levels of the intracellular ATP content ( H ), the extracellular lactic acid secretion ( I ), and the mRNA levels of glycolysis-related genes ( J ) were detected. The target gene transcription was normalized to ACTB . * P < 0.05 as compared with normal liver tissues group. # P < 0.05 as compared with Huh7 group. & P < 0.05 as compared with HBx-expressing Huh7 group. pc3.1: pcDNA3.1 transfection without HBx-expressing. pc3.1-HBx: pcDNA3.1-HBx transfection with HBx-expressing.

Article Snippet: Human hepatocellular carcinoma Huh7 and MHCC-97H cell lines were obtaineded from ATCC and maintained in our laboratory.

Techniques: Derivative Assay, Infection, Expressing, Transfection, Cell Culture, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Dysregulation of miRISC Regulatory Network Promotes Hepatocellular Carcinoma by Targeting PI3K/Akt Signaling Pathway

doi: 10.3390/ijms231911300

Figure Lengend Snippet: The relative miR-221 and AEG-1 mRNA levels in HCC cell lines. The miR-221 ( A ) and AEG-1 ( B ) relative mRNA levels analyzed in HCC cell line panels, the relative miR-221 expression in miR-221 mimic- ( C ), miR-221 inhibitor- ( D ), and AEG-1 siRNA- ( E ) transfected HCC cells. The relative mRNA levels of AEG-1 in miR-221 mimic/inhibitor- ( F ) and AEG-1 siRNA-transfected groups in HCC cells ( G ). The RNU6 and GAPDH were used as internal controls. Error bars presented as mean ± s.d and p -values represented as *** p < 0.001, **** p < 0.0001. ns represented as non-significance.

Article Snippet: Human HCC cell lines (HepG2, Huh7, and Hep3B) were obtained from the National Centre for Cell Science (NCCS), Pune, India, and cultured in DMEM with 10% FBS (fetal bovine serum) and 1% PS (penicillin streptomycin) (Invitrogen, Carlsbad, CA, USA).

Techniques: Expressing, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Dysregulation of miRISC Regulatory Network Promotes Hepatocellular Carcinoma by Targeting PI3K/Akt Signaling Pathway

doi: 10.3390/ijms231911300

Figure Lengend Snippet: AEG-1 and miR-221 regulate angiogenesis and cell cycle regulatory mRNA expressions in the HCC cell lines. The regulatory mRNAs expressions which regulate angiogenesis (LSF and MMP9) and cell cycle (p57, p53, and RB) were analyzed in miR-221 mimic-, miR-221 inhibitor-, AEG-1 siRNA-, and their corresponding control-transfected HepG2, Huh7, and Hep3B cells by using qRT- PCR. The GAPDH was used as an internal control. Error bars presented as mean ± s.d and p -values represented as * p < 0.05, ** p < 0.01, and *** p < 0.001. ns represented as non-significance.

Article Snippet: Human HCC cell lines (HepG2, Huh7, and Hep3B) were obtained from the National Centre for Cell Science (NCCS), Pune, India, and cultured in DMEM with 10% FBS (fetal bovine serum) and 1% PS (penicillin streptomycin) (Invitrogen, Carlsbad, CA, USA).

Techniques: Control, Transfection, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Dysregulation of miRISC Regulatory Network Promotes Hepatocellular Carcinoma by Targeting PI3K/Akt Signaling Pathway

doi: 10.3390/ijms231911300

Figure Lengend Snippet: AEG-1/miR-221 regulates the apoptosis and autophagy regulatory mRNAs in the HCC cell lines. The apoptosis and autophagy regulatory mRNAs (OPN, Bcl-2, PTEN, LC3A, PI3K, and Akt) expressions were analyzed in miR-221 mimic-, miR-221 inhibitor, and AEG-1 siRNA-transfected HCC cells by using qRT-PCR, and GAPDH used as an internal control. Error bars presented as mean ± s.d. and p -value represented as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared with corresponding controls.

Article Snippet: Human HCC cell lines (HepG2, Huh7, and Hep3B) were obtained from the National Centre for Cell Science (NCCS), Pune, India, and cultured in DMEM with 10% FBS (fetal bovine serum) and 1% PS (penicillin streptomycin) (Invitrogen, Carlsbad, CA, USA).

Techniques: Transfection, Quantitative RT-PCR, Control

Journal: International Journal of Molecular Sciences

Article Title: Dysregulation of miRISC Regulatory Network Promotes Hepatocellular Carcinoma by Targeting PI3K/Akt Signaling Pathway

doi: 10.3390/ijms231911300

Figure Lengend Snippet: Silencing of AEG-1 and miR-221 inhibits HCC cell migration and proliferation in vitro. The effect of the miR-221/AEG-1 measured on HCC cell migration and cell proliferation in the miR-221 mimic-, miR-221 inhibitor-, AEG-1 siRNA-, and their control-transfected HepG2, Huh7, and Hep3B cells by invasion ( A ), migration ( B ), and MTT assay ( C ) in vitro. Images analyzed using Image J (NIH) (scale bar, 100 µm). Error bars presented as mean ± s.d and p -value represented as ** p < 0.01, *** p < 0.001 compared to the corresponding controls.

Article Snippet: Human HCC cell lines (HepG2, Huh7, and Hep3B) were obtained from the National Centre for Cell Science (NCCS), Pune, India, and cultured in DMEM with 10% FBS (fetal bovine serum) and 1% PS (penicillin streptomycin) (Invitrogen, Carlsbad, CA, USA).

Techniques: Migration, In Vitro, Control, Transfection, MTT Assay

Journal: International Journal of Molecular Sciences

Article Title: Dysregulation of miRISC Regulatory Network Promotes Hepatocellular Carcinoma by Targeting PI3K/Akt Signaling Pathway

doi: 10.3390/ijms231911300

Figure Lengend Snippet: The effects of AEG-1/miR-221 on HCC cell cycle regulation and apoptosis. The effects of AEG-1/miR-221 on apoptosis ( A ) and cell cycle ( B ) analyzed in the miR-221 mimic-, and miR-221 inhibitor-, AEG-1 siRNA-, and their corresponding control-transfected HCC cells in vitro by flow cytometry analysis. Error bars presented as the mean ± s.d. and p -values represented as ** p < 0.01, *** p < 0.001 compared to the corresponding controls.

Article Snippet: Human HCC cell lines (HepG2, Huh7, and Hep3B) were obtained from the National Centre for Cell Science (NCCS), Pune, India, and cultured in DMEM with 10% FBS (fetal bovine serum) and 1% PS (penicillin streptomycin) (Invitrogen, Carlsbad, CA, USA).

Techniques: Control, Transfection, In Vitro, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Dysregulation of miRISC Regulatory Network Promotes Hepatocellular Carcinoma by Targeting PI3K/Akt Signaling Pathway

doi: 10.3390/ijms231911300

Figure Lengend Snippet: The effect of AEG-1/miR-221 was analyzed on cell cycle and angiogenesis regulatory proteins in HCC cell lines. The relative AEG-1 protein expression in THLE-2, HepG2, Huh7, and Hep3B cells ( A ) and miR-221 mimic-, miR-221 inhibitor-, and AEG-1 siRNA-transfected HCC cells ( B ). The regulatory protein expressions which regulate angiogenesis (LSF and MMP 9) and cell cycle (p57, p53, and RB) were analyzed in miR-221 mimic-, miR-221 inhibitor-, AEG-1 siRNA-, and their controls-transfected HepG2 ( C ), Huh 7 ( D ), and Hep3B ( E ) cells by western blot. p -Values presented as * p < 0.05, ** p < 0.01,*** p < 0.001, **** p < 0.0001 and error bars presented as the mean ± s.d. ns—(non-significant) compared to the controls.

Article Snippet: Human HCC cell lines (HepG2, Huh7, and Hep3B) were obtained from the National Centre for Cell Science (NCCS), Pune, India, and cultured in DMEM with 10% FBS (fetal bovine serum) and 1% PS (penicillin streptomycin) (Invitrogen, Carlsbad, CA, USA).

Techniques: Expressing, Transfection, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Dysregulation of miRISC Regulatory Network Promotes Hepatocellular Carcinoma by Targeting PI3K/Akt Signaling Pathway

doi: 10.3390/ijms231911300

Figure Lengend Snippet: Ectopic expression of AEG-1/miR-221 dysregulates apoptosis and autophagy regulatory protein levels in HCC cell lines. The relative expressions of apoptosis (OPN and Bcl-2), autophagy (LC3A/B), PTEN, and PI3K/Akt proteins were analyzed by western blotting in the mock control, miR-221 mimic, miR-221 inhibitor, AEG-1 siRNA, and corresponding controls transfected HepG2 ( A ), Huh7 ( B ), and Hep3B ( C ) cells using β-actin as an internal control. Error bars are represented as mean ± s.d. and * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to the control group. ns: non-significance.

Article Snippet: Human HCC cell lines (HepG2, Huh7, and Hep3B) were obtained from the National Centre for Cell Science (NCCS), Pune, India, and cultured in DMEM with 10% FBS (fetal bovine serum) and 1% PS (penicillin streptomycin) (Invitrogen, Carlsbad, CA, USA).

Techniques: Expressing, Western Blot, Control, Transfection

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Overexpression of galectin-4 promotes cell growth of hepatocellular carcinoma cells in vitro and in vivo

doi:

Figure Lengend Snippet: Galectin-4 inhibited HCC cell apoptosis by regulation of Bax and Bcl-2 in vitro. A. Flow cytometry was used to analyze the cell apoptosis in HCCLM3 cells with stable galectin-4 overexpression/knockdown. B. Western blotting analysis of Bax and Bcl-2 in HCCLM3 cells with stable galectin-4 overexpression/knockdown. *P<0.05.

Article Snippet: Human HCC cells (HepG2, Huh-7, and sk-hep-1) were purchased from the American Type Culture Collection.

Techniques: In Vitro, Flow Cytometry, Over Expression, Knockdown, Western Blot

Journal: Chinese Medical Journal

Article Title: USP25 promotes hepatocellular carcinoma progression by interacting with TRIM21 via the Wnt/β-catenin signaling pathway

doi: 10.1097/CM9.0000000000002714

Figure Lengend Snippet: USP25 is highly expressed in HCC and is correlated with poor prognosis. (A) The expression of USP25 in pan-carcinoma as determined with the TIMER database. (B) The expression of USP25 mRNA was analyzed in HCC tissues from TCGA and ICGC databases. (C) The protein expression levels of USP25 in a normal liver cell line and six HCC cell lines. (D) The expression of USP25 in different HCC cell lines in the CCLE database. (E) Kaplan–Meier survival analysis of survival in HCC patients grouped according to USP25 expression ( n = 370). (F) The promoter methylation level of USP25 in different grades of HCC according to the analysis of patient samples. Grade 1: well differentiated; Grade 2: moderately-differentiated; Grade 3: moderately and poorly differentiated; Grade 4: poorly differentiated. (G) The expression of USP25 in HCC patients based on nodal metastasis status. * P <0.05; † P <0.001; ‡ P <0.01, § P <0.0001. ACC: Adrenocortieal carcinoma; BLCA: Bladder urothelial carcinoma; BRCA:Breast invasive carcinoma; CESC: Cervical squamous cell carcinoma; CHOL: Cholangiocarcinoma; COAD: Colon adenocarcinoma; DLBC: Lymphoid neoplasm diffuse large B-cell lymphoma; ESCA: Esophageal carcinoma; GBM: Glioblastoma multiforme; HNSC: head and neck squamous cell carcinoma; KICH: Kidney chromophobe; KIRC: Kidney renal clear cell carcinoma; KIRP: Kidney renal papillary cell carcinoma; LAML: Acute myeloid leukemia; LGG: Brain lower grade glioma; LIHC: Liver hepatocellular carcinoma; LUAD: Lung adenocarcinoma; LUSC: Lung squamous cell carcinoma; MESO: Mesothelioma; OV: Ovarian serous cystadenocarcinoma; PAAD: Pancreatic adenocarcinoma; PCPG: Pheochromocytoma and paraganglioma; PRAD: Prostate adenocarcinoma; READ: Rectum adenocarcinoma; SARC: Sarcoma; SKCM: Skin cutaneous melanoma; STAD: Stomach adenocarcinoma; TGCT: Testicular germ cell tumors; THCA: Thyroid carcinoma; THYM: Thymoma; UCEC: Uterine corpus endometrial carcinoma; UCS: Uterine carcinosarcoma; UVM: Uveal melanoma; HCC: Hepatocellular carcinoma; ICGC: International Cancer Genome Consortium; TIMER: Tumor Immune Estimation Resource; USP25: Ubiquitin-specific peptidase 25; TCGA: The Cancer Genome Atlas; CCLE: Cancer Cell Line Encyclopedia; HR: Hazard ratio; L-O2: Normal hepatocyte cell line; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; N0, 1: Node0,1.

Article Snippet: Six human HCC cell lines (HepG2, HuH7, SMMC7721, Bel7402, MHCC97H, and PLC/PRF/5) and a normal hepatocyte cell line (L-O2) were obtained from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Expressing, Methylation, Ubiquitin Proteomics

Journal: Chinese Medical Journal

Article Title: USP25 promotes hepatocellular carcinoma progression by interacting with TRIM21 via the Wnt/β-catenin signaling pathway

doi: 10.1097/CM9.0000000000002714

Figure Lengend Snippet: USP25 promotes HCC cell proliferation in vitro. (A–C) HepG2 and MHCC97H cell lines with USP25 overexpression or knockdown were successfully established, as verified by analysis of protein and mRNA levels and fluorescence microscopy (Original magnification × 100). (D–F) Colony formation and CCK-8 assays were performed to detect cell proliferation. (G–J) DNA synthesis was measured by EdU IF staining (Original magnification × 200). * P <0.05; † P <0.001; ‡ P <0.01.CCK-8: Cell counting kit-8; HCC: Hepatocellular carcinoma; IF: Immunofluorescence; USP25: Ubiquitin-specific peptidase 25; EdU: 5-ethnyl-2-deoxyuridine; DAPI: 4′,6-Diamidino-2′-phenylindole; shUSP25: short hairpin Ubiquitin-specific peptidase 25; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Six human HCC cell lines (HepG2, HuH7, SMMC7721, Bel7402, MHCC97H, and PLC/PRF/5) and a normal hepatocyte cell line (L-O2) were obtained from the China Center for Type Culture Collection (Wuhan, China).

Techniques: In Vitro, Over Expression, Knockdown, Fluorescence, Microscopy, CCK-8 Assay, DNA Synthesis, Staining, Cell Counting, Immunofluorescence, Ubiquitin Proteomics

Journal: Chinese Medical Journal

Article Title: USP25 promotes hepatocellular carcinoma progression by interacting with TRIM21 via the Wnt/β-catenin signaling pathway

doi: 10.1097/CM9.0000000000002714

Figure Lengend Snippet: USP25 promotes HCC cell migration and invasion in vitro. (A,B) A Transwell invasion assay was performed to assess the invasiveness of HepG2 and MHCC97H cells (Original magnification × 100). (C,D) A transwell migration assay was performed to analyze the migratory ability of HepG2 and MHCC97H cells (Original magnification × 100). (E–H) The scratch assay results demonstrated the migratory ability of HepG2 and MHCC97H cells (Original magnification × 40). (I,J) The expression levels of a PCNA and invasion-related proteins (MMP2 and MMP9) were detected by Western blotting. * P <0.001; † P <0.01. HCC: Hepatocellular carcinoma; PCNA: Proliferation-related protein; USP25: Ubiquitin-specific peptidase 25; MMP2: Matrix metallopeptidase 2; MMP9: Matrix metallopeptidase 9; shUSP25: short hairpin Ubiquitin-specific peptidase 25; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Six human HCC cell lines (HepG2, HuH7, SMMC7721, Bel7402, MHCC97H, and PLC/PRF/5) and a normal hepatocyte cell line (L-O2) were obtained from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Migration, In Vitro, Transwell Invasion Assay, Transwell Migration Assay, Wound Healing Assay, Expressing, Western Blot, Ubiquitin Proteomics

Journal: Chinese Medical Journal

Article Title: USP25 promotes hepatocellular carcinoma progression by interacting with TRIM21 via the Wnt/β-catenin signaling pathway

doi: 10.1097/CM9.0000000000002714

Figure Lengend Snippet: USP25 promotes HCC growth by activating the Wnt/β-catenin signaling pathway. (A) The expression of β-catenin was observed by IF staining (Original magnification × 200). (B) The mRNA expression of components in the Wnt/β-catenin signaling pathway was measured by qRT-PCR. (C) The expression of β-catenin was observed by Western blotting of nuclear-cytoplasmic fractionation. (D, E) Western blotting was used to detect the protein expression levels of Wnt/β-catenin pathway components and downstream factors. (F, G) Western blotting analysis after XAV939 treatment of HepG2 and MHCC97H cells. * P <0.001; † P <0.01; ‡ P <0.05, shUSP25 vs . Control; § P <0.05, shUSP25+XAV939 vs . Control. HCC: Hepatocellular carcinoma; IF: Immunofluorescence; qRT-PCR: Quantitative real-time polymerase chain reactio; USP25: Ubiquitin-specific peptidase 25.DAPI: 4′,6-Diamidino-2′-phenylindole; shUSP25: Short hairpin Ubiquitin-specific peptidase 25; PCNA: Proliferating cell nuclear antigen GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; GSK3β: Glycogen synthase kinase 3 beta.

Article Snippet: Six human HCC cell lines (HepG2, HuH7, SMMC7721, Bel7402, MHCC97H, and PLC/PRF/5) and a normal hepatocyte cell line (L-O2) were obtained from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Expressing, Staining, Quantitative RT-PCR, Western Blot, Fractionation, Control, Immunofluorescence, Ubiquitin Proteomics