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: Scientific Reports

Article Title: Molecular basis of the interaction of the human tyrosine phosphatase PTPN3 with the hepatitis B virus core protein

doi: 10.1038/s41598-020-79580-9

Figure Lengend Snippet: Effects of PTPN3 overexpression on HBV life cycle. ( a ) PTPN3 expression in HepG2 NTCP or HepG2 NTCP-PTPN3 cells was tested by RT-qPCR. ( b ) HepG2 NTCP or HepG2 NTCP-PTPN3 cells were infected at a multiplicity of infection (MOI) of 100 and 7 days after infection, effect of PTPN3 expression was analyzed: (left panel) cccDNA levels were analyzed by qPCR after nucleocytoplasmic fractioning; (second panel) HBV total RNA was analyzed and quantified by RT-qPCR; (third panel) cytoplasmic HBV DNA was quantified by qPCR after nucleocytoplasmic fractionation; (right panel) secreted HBV DNA was quantified by qPCR after preS1 immunoprecipitation. Results are expressed as the mean of six independent experiments, and error bars represent standard error of the mean (SEM). p values were determined by Wilcoxon matched paired test * p < 0.05.

Article Snippet: HepG2-NTCP-PTPN3 cells are derived from HepG2-NTCP cells, and stably express PTPN3. pCMV6-PTPN3 plasmid was from ORIGENE (reference RC15851).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Infection, Fractionation, Immunoprecipitation

Journal: Antioxidants

Article Title: Myrobalan Fruit Extracts Modulate Immunobiochemical Pathways In Vitro

doi: 10.3390/antiox14030350

Figure Lengend Snippet: Effects of BB3 and the fruit extracts on cell viability. HepG2 cells were treated with vehicle (EtOH) or BB3 or myrobalan extracts (12.5–200 μg/mL) for 24 ( A ), 48 ( B ), and 72 h ( C ). Mean values ± S.E.M. of four independent experiments run in duplicates (* p < 0.05 and ** p < 0.005; compared to untreated cells) are shown.

Article Snippet: The human liver cancer-derived cell line HepG2 (RRID:CVCL_0027; DSMZ, Germany) was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco—Thermo Fisher Scientific, Vienna, Austria) supplemented with 10% ( v / v ) heat-inactivated FCS.

Techniques:

Journal: Antioxidants

Article Title: Myrobalan Fruit Extracts Modulate Immunobiochemical Pathways In Vitro

doi: 10.3390/antiox14030350

Figure Lengend Snippet: Measurement of intracellular ROS. Inhibition of peroxyl-radical (AAPH)-induced formation of ROS in HepG2 cells pretreated with increasing concentrations of BB3 or the myrobalan extracts (12.5–200 μg/mL). The mean percentages of DCF fluorescence, as a measure of ROS formation, are shown in relation to the AAPH-treated EtOH vehicle control (set to 100%). Mean values ± S.E.M. of three independent experiments run in quadruplicates (** p < 0.01; compared to AAPH-treated cells) are shown.

Article Snippet: The human liver cancer-derived cell line HepG2 (RRID:CVCL_0027; DSMZ, Germany) was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco—Thermo Fisher Scientific, Vienna, Austria) supplemented with 10% ( v / v ) heat-inactivated FCS.

Techniques: Inhibition, Fluorescence, Control

Journal: Antioxidants

Article Title: Myrobalan Fruit Extracts Modulate Immunobiochemical Pathways In Vitro

doi: 10.3390/antiox14030350

Figure Lengend Snippet: Influence of plant extracts on ß-lactamase activity, which was taken as a measure for ARE-dependent gene expression, in CellSensor ® ARE-bla HepG2 cells (expressed as n-fold to the corresponding control solvent). Shown are the mean values ± S.E.M. of three independent experiments with four parallels per concentration. (* p < 0.05 and ** p < 0.01; compared to vehicle-treated control cells).

Article Snippet: The human liver cancer-derived cell line HepG2 (RRID:CVCL_0027; DSMZ, Germany) was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco—Thermo Fisher Scientific, Vienna, Austria) supplemented with 10% ( v / v ) heat-inactivated FCS.

Techniques: Activity Assay, Gene Expression, Control, Solvent, Concentration Assay

Journal: Toxicology and Applied Pharmacology

Article Title: Remdesivir; molecular and functional measures of mitochondrial safety

doi: 10.1016/j.taap.2021.115783

Figure Lengend Snippet: Remdesivir Time- and Dose-dependent Cytotoxicity – HepG2/C3A cells were exposed to remdesivir for up to 72 h in 24-well plates containing increasing μM concentrations of drug. At the end of the exposure the cells were fixed with methanol and stained with sulphorhodamine B (SRB). The bound SRB was solubilized in 10 mM Tris base and the absorbance measured 540 nm. Error bars reflect the standard deviation of duplicate wells ( n = 1) for each concentration of remdesivir.

Article Snippet: Human-derived HepG2 liver cells were exposed for up to 48 h in culture to increasing concentrations of remdesivir.

Techniques: Staining, Standard Deviation, Concentration Assay

Journal: Toxicology and Applied Pharmacology

Article Title: Remdesivir; molecular and functional measures of mitochondrial safety

doi: 10.1016/j.taap.2021.115783

Figure Lengend Snippet: Dose-dependent Effect of Remdesivir on Mitochondrial DNA Copy Number in HepG2 Cells – A) HepG2 cells were exposed to increasing μM concentrations of remdesivir for 24 or 48 h. At the end of the exposure, DNA was extracted and quantified by qPCR using the nuclear (nDNA) and mitochondrial (mtDNA) gene targets recommended by Malik at el. (2011). B) HepG2 cells were exposed to sub-cytotoxic concentration of 2′, 3′ –dideoxycytidine (40 nM ddC) for 48 h as a positive control for mtDNA depletion. The values are expressed as mtDNA/nDNA copies and expressed as the mean ± S.D. for 3 independent measures. Asterisks indicate statistically significant difference from vehicle control (Student's t -test P < 0.05).

Article Snippet: Human-derived HepG2 liver cells were exposed for up to 48 h in culture to increasing concentrations of remdesivir.

Techniques: Concentration Assay, Positive Control

Journal: Toxicology and Applied Pharmacology

Article Title: Remdesivir; molecular and functional measures of mitochondrial safety

doi: 10.1016/j.taap.2021.115783

Figure Lengend Snippet: Dose-dependent Effect of Remdesivir on Mitochondrial and Nuclear Gene Expression. Panels A and C–H) HepG2 cells were exposed in culture to increasing concentrations of remdesivir for 24 and 48 h. RNA was extracted and RT-qPCR performed to quantify gene transcripts. Values are mRNA copies normalized to 18 s ribosomal RNA. B) HepG2 cells were exposed to a sub-toxic concentration of 2′- C -methyladeonsoine (25 μM 2-C-MA) as a positive control for mitochondrial RNA polymerase inhibition. Values are normalized to 18 s RNA and expressed as a percent of control. The plots represent the mean ± S.D. for 3 independent replications and asterisks indicate a statistically significant difference compared to the 0 μM dose group (Dunnett's p < 0.05).

Article Snippet: Human-derived HepG2 liver cells were exposed for up to 48 h in culture to increasing concentrations of remdesivir.

Techniques: Expressing, Quantitative RT-PCR, Concentration Assay, Positive Control, Inhibition

Journal: Toxicology and Applied Pharmacology

Article Title: Remdesivir; molecular and functional measures of mitochondrial safety

doi: 10.1016/j.taap.2021.115783

Figure Lengend Snippet: Cellular Oxygen Consumptions Parameters Associated with Remdesivir Exposure. Oxygen consumption rates (OCR) were measured for HepG2 cells exposed to increasing concentrations of remdesivir for 48 h. Each panel represents a parameter calculated according to the formula listed in and each bar reflects the mean ± S.D. for 4 independent experimental observations. Asterisks (*) indicate a statistically significant difference compared to the zero remdesivir control (Dunnett's, p ≤ 0.05).

Article Snippet: Human-derived HepG2 liver cells were exposed for up to 48 h in culture to increasing concentrations of remdesivir.

Techniques: