Journal: Lipids in Health and Disease

Article Title: The mechanism of apoliprotein A1 down-regulated by Hepatitis B virus

doi: 10.1186/s12944-016-0232-5

Figure Lengend Snippet: Suppression of ApoA1 expression by HBV. a and b ApoA1 mRNA and protein levels were detected by RT-PCR and Western blot in HepG2.2.15 corresponding HepG2 cell lines. c and d HepG2 cells were transfected with 2 μg pHBV1.3 plasmid or 2 μg pCDNA3.1 as control, ApoA1 mRNA and protein levels were detected at 48 h after transfection. Data are presented as the mean ± SD from three independent experiments. * p < 0.05 and ** P < 0.01 compared with mock

Article Snippet: Human hepatocellular carcinoma HepG2 and HepG2.2.15 cell lines were obtained from the ATCC (Rockville, MD), HepG2.2.15 with abilities to produce HBV virus stably is derived from HepG2 cell lines [ ].

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Plasmid Preparation, Control

Journal: Lipids in Health and Disease

Article Title: The mechanism of apoliprotein A1 down-regulated by Hepatitis B virus

doi: 10.1186/s12944-016-0232-5

Figure Lengend Snippet: ApoA1 expression was suppressed by DNA methyltransferase inhibitor 5-aza-dC. a and b 5 CpG islands including two methylation CpG status in ApoA1 promotor were listed ( a ), the detection results of the 5 CpG islands methylation status were shown ( b ). HepG2.2.15 cells treated with 5 μM 5-aza-dC 48 h, ApoA1 mRNA and protein levels were detected by RT-PCR and Western blot respectively ( c and d ). Secretion of HBsAg and HBV particles in the supernatant were detected by ELISA and RT- PCR respectivly ( e and f ). Data are presented as the mean ± SD from three independent experiments. * p < 0.05 and ** P < 0.01 compared with mock

Article Snippet: Human hepatocellular carcinoma HepG2 and HepG2.2.15 cell lines were obtained from the ATCC (Rockville, MD), HepG2.2.15 with abilities to produce HBV virus stably is derived from HepG2 cell lines [ ].

Techniques: Expressing, Methylation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Lipids in Health and Disease

Article Title: The mechanism of apoliprotein A1 down-regulated by Hepatitis B virus

doi: 10.1186/s12944-016-0232-5

Figure Lengend Snippet: Decreased HBV expression with 5-aza-dC treatment via up-regulation of ApoA1 expression. a ApoA1 overexpression inversely suppressed HBV expression. HepG2 cells were cotransfected with 1 μg pApoA1 plasmid and 1 μg pHBV1.3. Sectetion of HBsAg and HBeAg were analyzed by ELISA at 48 h after transfection. b The inhibitory effect of 5-aza-dC on HBV expression was completely abolished by blocking 5-aza-dC-induced up-regulation of ApoA1 using RNAi. HepG2.2.15 cells were treated with 5 μM 5-aza-dC plus 50 μM ApoA1 siRNA or negative control, expression of HBsAg and HBeAg in the supernatant were analyzed by ELISA at 48 h. Data are presented as the mean ± SD from three independent experiments. * p < 0.05 and ** P < 0.01 compared with mock

Article Snippet: Human hepatocellular carcinoma HepG2 and HepG2.2.15 cell lines were obtained from the ATCC (Rockville, MD), HepG2.2.15 with abilities to produce HBV virus stably is derived from HepG2 cell lines [ ].

Techniques: Expressing, Over Expression, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Transfection, Blocking Assay, Negative Control

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: Journal of King Saud University - Science

Article Title: Cytotoxic potential of Commicarpus plumbagineus extracts against liver cancer cell lines through In-Vitro and In-Silico methods

doi: 10.1016/j.jksus.2024.103253

Figure Lengend Snippet: Fig. 2. The cytotoxicity of the F2 extract from C. plumbagineus was evaluated on human liver cancer cells (HepG2 and HuH7) and non-cancerous cells (Huvec) using various concentrations (0–700 µg/mL) for a 24-hour treatment. Cell viability was assessed through the MTT assay, and statistical analysis was carried out utilising Student’s t-test. The data, presented as the mean ± stan dard deviation, were derived from three replicates. Significance was established at *p < 0.05 in comparison to the control group.

Article Snippet: Human hepatocellular carcinoma cells HuH7 and HepG2 and human umbilical vein endothelial non-cancerous cell line (HUVECs) (DSMZ, Germany) were maintained in DMEM (Gibco) supplemented with fetal R. Alghamdi et al. Journal of King Saud University - Science 36 (2024) 103253 bovine serum (FBS 10 %) (Gibco), 10 mM L-glutamine, at 37 ◦C in CO2 incubator.

Techniques: MTT Assay, Derivative Assay, Comparison, Control