hbv replication  (R&D Systems)

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Comprehensive Proteomics Identification of IFN-λ3-regulated Antiviral Proteins in HBV-transfected Cells *

doi: 10.1074/mcp.RA118.000735

Figure Lengend Snippet: Effects of IFN-λ3 on HBV replication. HepG2.2.15 cells were incubated with IFN-λ3 (1, 10, 100 and 1000 ng/ml) or treated with PBS for 24 h. The relative transcript expression and the amount of intracellular HBV DNA are shown as Mean ± S.E. IFN-λ3 significantly inhibited preS1 and pgRNA expression at doses equal to or greater than 10 ng/ml (A and B). IFN-λ3 significantly suppressed viral propagation at doses equal to or greater than 100 ng/ml (C). These experiments were performed in triplicate. (* represents a p value less than 0.05 and ** represents a p value less than 0.01).

Article Snippet: One million HepG2.2.15 cells were seeded into 6-well plates with 1 ml media and maintained in complete DMEM for 24 h. For determining the effects of IFN-λ3 on HBV replication, these cells were left untreated or treated with 1, 10, 100 or 1000 ng/ml of IFN-λ3 (5259-IL-025, R&D Systems, MN) and incubated for another 24 h. For proteomics analysis, HepG2.2.15 cells were plated at 5 × 10 6 cells in T-75 flasks and grown in complete DMEM for 24 h at 37 °C.

Techniques: Incubation, Expressing

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Comprehensive Proteomics Identification of IFN-λ3-regulated Antiviral Proteins in HBV-transfected Cells *

doi: 10.1074/mcp.RA118.000735

Figure Lengend Snippet: Illustration of HBV life-cycle mapped to antiviral proteins that were identified in this study. Antiviral proteins and host cellular factors of interest identified in this study were mapped into the HBV-life cycle. These proteins were identified at most steps of HBV replication. Proteins in red and green text represent up- and down-regulated proteins, respectively.

Article Snippet: One million HepG2.2.15 cells were seeded into 6-well plates with 1 ml media and maintained in complete DMEM for 24 h. For determining the effects of IFN-λ3 on HBV replication, these cells were left untreated or treated with 1, 10, 100 or 1000 ng/ml of IFN-λ3 (5259-IL-025, R&D Systems, MN) and incubated for another 24 h. For proteomics analysis, HepG2.2.15 cells were plated at 5 × 10 6 cells in T-75 flasks and grown in complete DMEM for 24 h at 37 °C.

Techniques:

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Comprehensive Proteomics Identification of IFN-λ3-regulated Antiviral Proteins in HBV-transfected Cells *

doi: 10.1074/mcp.RA118.000735

Figure Lengend Snippet: IFN-λ3 rescued the RIG-I signaling pathway. IFN-λ3 up-regulated the expression of RIG-I and IFIT3, which have been reported to be suppressed by HBV. IFN-λ3 also elevated the expression of OASL and TRIM25. These proteins promote type I and type III IFN production. These IFNs, in turn, activate the JAK-STAT pathway and induce the expression of ISGs. It is likely, then, that IFN-λ3 provides positive feedback to amplify ISG expression to control HBV replication. Proteins in red text represent up-regulated proteins, whereas the blue text represents the up-regulated transcripts.

Article Snippet: One million HepG2.2.15 cells were seeded into 6-well plates with 1 ml media and maintained in complete DMEM for 24 h. For determining the effects of IFN-λ3 on HBV replication, these cells were left untreated or treated with 1, 10, 100 or 1000 ng/ml of IFN-λ3 (5259-IL-025, R&D Systems, MN) and incubated for another 24 h. For proteomics analysis, HepG2.2.15 cells were plated at 5 × 10 6 cells in T-75 flasks and grown in complete DMEM for 24 h at 37 °C.

Techniques: Expressing, Control

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Comprehensive Proteomics Identification of IFN-λ3-regulated Antiviral Proteins in HBV-transfected Cells *

doi: 10.1074/mcp.RA118.000735

Figure Lengend Snippet: Outcome tree. To compare the effects of IFN-λ3, IFN-α2a, and PBS control, we generated a tree representing the 27 possible outcomes. The boxes in red and green represent proteins that are significantly up- and down-regulated, respectively. Groups of proteins with insignificantly changed expression are represented by yellow boxes. As an example, the topmost outcome represents proteins in which IFN-λ3 treatment causes significant up-regulation versus control, IFN-α2a treatment causes significant up-regulation versus control, and IFN-λ3 treatment causes significant up-regulation versus IFN-α2a. The number in parentheses refers to the number of identified proteins in the group. The proteins in each group that might be involved in suppressing HBV replication are shown in colored boxes specifying various processes, particularly antiviral.

Article Snippet: One million HepG2.2.15 cells were seeded into 6-well plates with 1 ml media and maintained in complete DMEM for 24 h. For determining the effects of IFN-λ3 on HBV replication, these cells were left untreated or treated with 1, 10, 100 or 1000 ng/ml of IFN-λ3 (5259-IL-025, R&D Systems, MN) and incubated for another 24 h. For proteomics analysis, HepG2.2.15 cells were plated at 5 × 10 6 cells in T-75 flasks and grown in complete DMEM for 24 h at 37 °C.

Techniques: Control, Generated, Expressing

Journal: Pharmaceuticals

Article Title: Potential Benefits of In Silico Methods: A Promising Alternative in Natural Compound’s Drug Discovery and Repurposing for HBV Therapy

doi: 10.3390/ph18030419

Figure Lengend Snippet: Comprehensive table summarizing drug repurposing and natural compounds in HBV therapy through in silico methods.

Article Snippet: , Natural alkaloid exhibiting antimicrobial, anti-inflammatory, antidiabetic, and cholesterol-reducing effects, frequently utilized to promote metabolic and cardiovascular well-being. , HBV DNA Replication , Interferes with viral replication by targeting HBV DNA synthesis , Virtual screening, molecular dynamics , Potent inhibition of HBV polymerase; synergistic potential with antivirals , Preclinical stage , [ ] .

Techniques: In Silico, Reverse Transcription, Binding Assay, Virus, Inhibition, Infection, DNA Synthesis, Drug discovery, Activity Assay, Biomarker Discovery, Derivative Assay, Blocking Assay

Journal: Antiviral research

Article Title: Screening of an epigenetic compound library identifies BRD4 as a potential antiviral target for hepatitis B virus covalently closed circular DNA transcription.

doi: 10.1016/j.antiviral.2023.105552

Figure Lengend Snippet: Fig. 1. The screening paradigm for identification of cccDNA epigenetic inhibitors. (A) Schematic diagram of cccDNA-dependent HA-tagged HBeAg (HA-HBeAg) reporter cell line HepBHAe82. See text and previous publication for details (Cai et al., 2016). (B) Cayman epigenetic compound library consisting of 146 compound was subjected to screening in HepBHAe82 cells. (C) HepBHAe82 cells in 96-well-plate were induced for HBV replication and cccDNA accumulation in Tet-free medium for 14 days, followed by treatment with 3TC (10 μM) and each library compound (10 μM) in the presence of Tet, DMSO served as mock control. The treatment was refreshed once on day 2, and the supernatant was collected on day 4 for HA-HBeAg CLIA. (D) HepHA-HBe4 cell line constitutively expressing HA-HBeAg served as a counter screen to exclude compounds directly targeting HBeAg metabolism or secretion. Cells were treated with DMSO solvent control or library compounds every other day for 4 days, followed by HA-HBeAg CLIA. (E) Compounds that reduce HA-HBeAg in HepBHAe82 cells by more than 50% without inhibiting HA-HBeAg production in HepHA-HBe4 cells will be selected as primary hits.

Article Snippet: BRD4 degrader MZ1 (cat# S8889) and HBV replication inhibitor lamivudine (3TC) (cat# S1706) were purchased from Selleck Chemicals.

Techniques: Drug discovery, Control, Expressing, Solvent

Journal: Antiviral research

Article Title: Screening of an epigenetic compound library identifies BRD4 as a potential antiviral target for hepatitis B virus covalently closed circular DNA transcription.

doi: 10.1016/j.antiviral.2023.105552

Figure Lengend Snippet: Fig. 3. Primary hits validation. Primary hits gener ated from HepBHAe82 screening assay were further validated in HepAD38 cell line. HepAD38 cells were cultured in Tet-free medium to induce HBV replica tion and cccDNA synthesis for 10 days, and then treated with 10 μM of primary hit compounds every other day for 4 days in the presence of Tet and 3TC (10 μM), DMSO served as solvent mock treatment control. (A) HBV Hirt DNA was extracted and sub jected to cccDNA quantification by qPCR. Relative cccDNA levels are plotted as fold change to DMSO control. (B) Total RNA was extracted and subjected to HBV pC mRNA qPCR. Relative pC mRNA levels are plotted as fold change to DMSO control. (C) The relative levels of cccDNA-based transcription were further determined by normalizing pC mRNA level to cccDNA level in each treatment group compared to DMSO control group. (D) The validated hits UNC0642, CAY10433, and MS436 were repurchased and their antiviral effect on cccDNA transcription were further verified in HepBHAe82 cells by HA- HBeAg CLIA assay as described above. RLU: relative luminescence units. All data are shown as mean ± SD (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: BRD4 degrader MZ1 (cat# S8889) and HBV replication inhibitor lamivudine (3TC) (cat# S1706) were purchased from Selleck Chemicals.

Techniques: Biomarker Discovery, Screening Assay, Cell Culture, Solvent, Control

Journal: Antiviral research

Article Title: Screening of an epigenetic compound library identifies BRD4 as a potential antiviral target for hepatitis B virus covalently closed circular DNA transcription.

doi: 10.1016/j.antiviral.2023.105552

Figure Lengend Snippet: Fig. 4. Antiviral assessment of validated hits in HBV infection system. HepG2-NTCP cell were infected with HBV at 250 vge/cell for 3 days, followed by treatment with DMSO control, compound UNC0642, CAY10433 or MS436 at 10 μM for 4 days in the presence of 10 μM 3TC. HBV cccDNA-based gene expression was assessed by HBc immunofluorescence, the average percentage of HBc-positive cells were calculated from five microscope field of view. Nuclei were stained with DAPI. Scale bar: 50 μm.

Article Snippet: BRD4 degrader MZ1 (cat# S8889) and HBV replication inhibitor lamivudine (3TC) (cat# S1706) were purchased from Selleck Chemicals.

Techniques: Infection, Control, Gene Expression, Immunofluorescence, Microscopy, Staining

Journal: Antiviral research

Article Title: Screening of an epigenetic compound library identifies BRD4 as a potential antiviral target for hepatitis B virus covalently closed circular DNA transcription.

doi: 10.1016/j.antiviral.2023.105552

Figure Lengend Snippet: Fig. 5. CC50/EC50 of MS436. (A) The chemical structure of compound MS436. (B) CC50 measurement of MS436. HepG2 cell were treated with 2-fold serially diluted MS436 (0 μM–100 μM) every other day for 4 days, cell survival rate (% survival) was detected by MTT assay and plotted as percentage of DMSO con trol. The CC50 value was calculated through plotting % survival versus log concentration of MS436 via variable slope linear regression. (C) Determining EC50 of MS436. A range of experimental concentrations (0 μM–200 μM, 2-fold serial dilutions) of MS436 were used to treat HepBHAe82 cells after induction for 14 days. The furin inhibitor I (1 μM) which can efficiently block the secretion of HA-HBeAg, served as control for background signal. Treatment was repeated every other day for 4 days in the presence of Tet and 3TC (10 μM). Supernatant samples were collected at treatment endpoint and subjected to HA- HBeAg CLIA analysis. HA-HBeAg CLIA signals were normalized by background signal and plotted as percentage of DMSO control. The EC50 was calculated through plotting % HA-HBeAg CLIA versus log concentration of MS436 via variable slope linear regression.

Article Snippet: BRD4 degrader MZ1 (cat# S8889) and HBV replication inhibitor lamivudine (3TC) (cat# S1706) were purchased from Selleck Chemicals.

Techniques: MTT Assay, Concentration Assay, Blocking Assay, Control

Journal: Antiviral research

Article Title: Screening of an epigenetic compound library identifies BRD4 as a potential antiviral target for hepatitis B virus covalently closed circular DNA transcription.

doi: 10.1016/j.antiviral.2023.105552

Figure Lengend Snippet: Fig. 6. Antiviral assessment of MS436 in HBV- infected HepG2-NTCP cells. HepG2-NTCP cells were infected with HBV (250 vge/cell) for 3 days, followed by treatment with DMSO solvent control and 10 μM MS436 every other day for 4 days in the presence of 10 μM 3TC. (A) HBV total RNA were extracted and detected by northern blot, 3.5 kb pC mRNA and pgRNA, 2.4/2.1 kb surface mRNAs were labeled. Ri bosomal RNA (rRNA) served as loading control. (B) HBV pC mRNA was detected by qPCR. The relative pC mRNA levels after MS436 treatment were plotted as fold change to DMSO control. (C) Hirt DNA was extracted, heat denatured and digested by PSAD to remove non-cccDNA species, followed by cccDNA qPCR. The relative cccDNA levels after MS436 treat ment were plotted as fold change to DMSO control. (D) The relative transcription activity of cccDNA was presented by normalizing pC mRNA level to cccDNA level and plotted as fold change to DMSO control group. Data are shown as mean ± SD (n = 3). *p < 0.05, ***p < 0.001.

Article Snippet: BRD4 degrader MZ1 (cat# S8889) and HBV replication inhibitor lamivudine (3TC) (cat# S1706) were purchased from Selleck Chemicals.

Techniques: Infection, Solvent, Control, Northern Blot, Labeling, Activity Assay

Journal: Antiviral research

Article Title: Screening of an epigenetic compound library identifies BRD4 as a potential antiviral target for hepatitis B virus covalently closed circular DNA transcription.

doi: 10.1016/j.antiviral.2023.105552

Figure Lengend Snippet: Fig. 8. MS436-mediated epigenetic change of cccDNA minichromosome. (A–D) HepAD38 cells cultured in T75 flask were induced in Tet-free me dium for 10 days for cccDNA establishment, then treated with DMSO or MS436 (10 μM) every other day for 4 days in the presence of 3TC (10 μM). Cells were crosslinked and lysed for further chromatin fragmentation by sonication-based shearing. cccDNA was immunoprecipitated by ChIP-grade non-immune IgG isotype control or antibodies against H3K27ac, H3K9me3, H3K4me3 or BRD4 and detected by qPCR. The enrichment of aforementioned histone PTMs or BRD4 protein on cccDNA was plotted as fold change to NIS IgG control, respectively. Data are shown as mean ± SD (n = 3). **p < 0.01, ***p < 0.001, ns: not significant. (E) The intracellular total BRD4 proteins, including both the long and short isoforms, were detected by western blot. β-actin served as the loading control.

Article Snippet: BRD4 degrader MZ1 (cat# S8889) and HBV replication inhibitor lamivudine (3TC) (cat# S1706) were purchased from Selleck Chemicals.

Techniques: Cell Culture, Sonication, Immunoprecipitation, Control, Western Blot

Journal: Antiviral research

Article Title: Screening of an epigenetic compound library identifies BRD4 as a potential antiviral target for hepatitis B virus covalently closed circular DNA transcription.

doi: 10.1016/j.antiviral.2023.105552

Figure Lengend Snippet: Fig. 9. Differential transcriptome analyses of MS436-treated HBV-infected cells. HepG2-NTCP cells were infected with HBV (250 vge/cell) for 3 days, followed by treatment with DMSO solvent control and 10 μM MS436 every other day for 4 days in the presence of 10 μM 3TC. The experiment was per formed in duplicate. Then, total RNA was extracted and subjected to RNAseq and the following Differential transcriptome analyses. (A) Volcano plot. The log2 Fold Change indicates the mean expression level change for each gene. Q- value indicates the FDR adjusted p-value for each comparison. Each dot rep resents one gene while red dots are significant DEGs (fold change >2 and Q- value <0.05) and black dots are insignificant DEGs. (B) Expression heatmap of significant DEGs. Expressions are normalized by the median of ratios method and then log-transformed for visualization. (C) Functional enrichment. Each row shows one function from Gene Ontology Biological Process. “Hits” in dicates the number of enriched genes in each term. Significance is converted from the p-value of each enrichment and colored from blue (less significant) to red (more significant).

Article Snippet: BRD4 degrader MZ1 (cat# S8889) and HBV replication inhibitor lamivudine (3TC) (cat# S1706) were purchased from Selleck Chemicals.

Techniques: Infection, Solvent, Control, Expressing, Comparison, Transformation Assay, Functional Assay

Journal: Antiviral research

Article Title: Screening of an epigenetic compound library identifies BRD4 as a potential antiviral target for hepatitis B virus covalently closed circular DNA transcription.

doi: 10.1016/j.antiviral.2023.105552

Figure Lengend Snippet: Fig. 12. Depletion of BRD4 by PROTAC degraders inhibits HBV transcription. (A, C) Chemical structure of dBET1 and MZ-1. (B, D) HepG2-NTCP cell were infected with HBV (500 vge/cell) for 6 days, followed by treatment with (B) dBET1 (10 μM) in the absence or presence of 3TC (10 μM) or (D) MZ-1 (1 μM) and 3TC (10 μM) every other day for 4 days. Cellular BRD4 was detected by western blot, β-actin served as loading control. HBV total RNA and/or cytoplasmic core DNA were detected by northern and Southern blot, respectively.

Article Snippet: BRD4 degrader MZ1 (cat# S8889) and HBV replication inhibitor lamivudine (3TC) (cat# S1706) were purchased from Selleck Chemicals.

Techniques: Infection, Western Blot, Control, Northern Blot, Southern Blot

Journal: PLoS Pathogens

Article Title: Hepatitis B virus X protein counteracts high mobility group box 1 protein-mediated epigenetic silencing of covalently closed circular DNA

doi: 10.1371/journal.ppat.1010576

Figure Lengend Snippet: The cell lines were seeded in 35mm-dish with density of 1.2×10 6 cells/dish and induced for HBV replication in the absence of tet for 16 days, and subjected to the following analyses: (A) Panels from top to bottom: HBV total viral RNA, including pregenomic (pg) RNA and surface mRNA (sRNA), were detected by Northern blot. Cellular 28S and 18S ribosomal RNA (rRNA) serving as RNA loading control; HBV cytoplasmic core DNA and HBV Hirt DNA were detected by Southern blot. The relaxed circular (RC) DNA, single stranded (SS) DNA, deproteinated RC (DP-RC) DNA and cccDNA are indicated; HBV pCore (pC) mRNA was detected by reverse transcription (RT)-PCR gel electrophoresis, cellular β-actin mRNA RT-PCR served as loading control. (B) cccDNA extracted from each cell lines in a 35-mm dish were quantified for copy numbers by qPCR. (C) Supernatant HA-HBeAg produced by the three cell lines at indicated time points were measured by CLIA and plotted as a histogram (Mean ± SD, n = 3).

Article Snippet: HBV replication inhibitor Lamivudine (3TC) was purchased from Selleck Chemicals and dissolved in DMSO as 10 mM stock.

Techniques: Northern Blot, Control, Southern Blot, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Produced

Journal: PLoS Pathogens

Article Title: Hepatitis B virus X protein counteracts high mobility group box 1 protein-mediated epigenetic silencing of covalently closed circular DNA

doi: 10.1371/journal.ppat.1010576

Figure Lengend Snippet: HepBHAeΔx67-shControl and HepBHAeΔx67-shHMGB1 cells were cultured in tet-free medium to induce HBV replication for 14 days, the association of (A) H3K27ac, (B) H3K27me3, and (C) RNAPII pho-CTD with cccDNA was analyzed by ChIP-qPCR and plotted in percentage (%) of input (mean ± SEM, n = 3). *p<0.05, ***p<0.001.

Article Snippet: HBV replication inhibitor Lamivudine (3TC) was purchased from Selleck Chemicals and dissolved in DMSO as 10 mM stock.

Techniques: Cell Culture, ChIP-qPCR

Journal: Journal of Translational Medicine

Article Title: Upregulation of microRNA-328-3p by hepatitis B virus contributes to THLE-2 cell injury by downregulating FOXO4

doi: 10.1186/s12967-020-02299-8

Figure Lengend Snippet: Effect of HBV/HBs/HBc/HBp/HBx on pro-inflammatory cytokines, miR-328-3p expression, STAT3 phosphorylation, and FOXO4 protein expression. THLE-2 cells were transfected with pHBV1.3 (HBV replication plasmid), pHBs (expressing HBsAg), pHBc (expressing HBcAg), pHBp (expressing DNA polymerase protein), pHBx (expressing HBx), and empty pcDNA3.1 vector (control), under LPS stimulation (1 μg/mL, 24 h). a ELISA was performed to detect levels of TNF-α, IL-6, IL-8, IL-12, and IL-18. b qRT-PCR was conducted to examine miR-328-3p expression. c Western blot was performed to measure protein expression of FOXO4 and STAT3, and phosphorylated (p)-STAT3. Values are presented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01 vs. NC. # P < 0.05, ## P < 0.01 vs. LPS + vector

Article Snippet: The HBV replication plasmid pHBV1.3 which contained 1.3 copies of the HBV genome was obtained from BioVector (Beijing, China).

Techniques: Expressing, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot

Journal: Journal of Translational Medicine

Article Title: Upregulation of microRNA-328-3p by hepatitis B virus contributes to THLE-2 cell injury by downregulating FOXO4

doi: 10.1186/s12967-020-02299-8

Figure Lengend Snippet: STAT3 activates miR-328-3p transcription and mediates the HBV/HBc/HBx-induced upregulation of miR-328-3p. a THLE-2 cells were cultured with recombinant human IL-6 protein (200 ng/mL), S3I-201 (50 μM), or DMSO (vehicle control) for 24 h, followed by qRT-PCR analysis of miR-328-3p expression. b Resultant ChIP DNA was amplified by qRT-PCR. CHIP-qPCR assay confirmed the direct binding of STAT3 to the miR-328 promoter. IgG served as a negative control. IL-6 was used to activate STAT3. c – e THLE-2 cells were transfected with pHBV1.3, pHBc, pHBx and empty pcDNA3.1 vector (control), followed by S3I-201 (50 μM, 24 h) or not, under LPS stimulation (1 μg/mL, 24 h). Then c qRT-PCR was conducted to examine miR-328-3p expression. d ELISA was performed to detect levels of TNF-α, IL-6, IL-8, IL-12, and IL-18. e Western blot was performed to measure protein expression of STAT3, and p-STAT3. Values are presented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01 vs. LPS + Vector. ## P < 0.01 vs. LPS + pHBV1.3. && P < 0.01 vs. LPS + pHBc. $$ P < 0.01 vs. LPS + pHBx

Article Snippet: The HBV replication plasmid pHBV1.3 which contained 1.3 copies of the HBV genome was obtained from BioVector (Beijing, China).

Techniques: Cell Culture, Recombinant, Quantitative RT-PCR, Expressing, Amplification, Binding Assay, Negative Control, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Journal of Translational Medicine

Article Title: Upregulation of microRNA-328-3p by hepatitis B virus contributes to THLE-2 cell injury by downregulating FOXO4

doi: 10.1186/s12967-020-02299-8

Figure Lengend Snippet: miR-328-3p inhibitor suppresses pHBV1.3-induced HBV activity. THLE-2 cells were co-transfected with pHBV1.3 or empty pcDNA3.1 vector (control), and miR-328-3p inhibitor or inhibitor negative control (NC). a HBV RNA and b HBV DNA were qualified. c , d The OD 450 absorbance values were obtained by ELISA for determining levels of HBsAg and HBeAg in THLE-2 cell supernatants. Values are presented as the mean ± SD (n = 3). ## P < 0.01 vs. Vector + inhibitor NC. && P < 0.01 vs. inhibitor NC + pHBV1.3

Article Snippet: The HBV replication plasmid pHBV1.3 which contained 1.3 copies of the HBV genome was obtained from BioVector (Beijing, China).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Negative Control, Enzyme-linked Immunosorbent Assay

Journal: Journal of Translational Medicine

Article Title: Upregulation of microRNA-328-3p by hepatitis B virus contributes to THLE-2 cell injury by downregulating FOXO4

doi: 10.1186/s12967-020-02299-8

Figure Lengend Snippet: FOXO4 suppresses pHBV1.3-induced HBV activity. THLE-2 cells were transfected with pHBV1.3 and pcDNA3.1-FOXO4, both alone and in combination. a HBV RNA and b HBV DNA were qualified. c The OD 450 absorbance values were obtained by ELISA for determining levels of HBsAg and HBeAg in THLE-2 cell supernatants. Values are presented as the mean ± SD (n = 3). **P < 0.01 vs. Vector. ## P < 0.01 vs. pHBV1.3

Article Snippet: The HBV replication plasmid pHBV1.3 which contained 1.3 copies of the HBV genome was obtained from BioVector (Beijing, China).

Techniques: Activity Assay, Transfection, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

Journal: Journal of Translational Medicine

Article Title: Upregulation of microRNA-328-3p by hepatitis B virus contributes to THLE-2 cell injury by downregulating FOXO4

doi: 10.1186/s12967-020-02299-8

Figure Lengend Snippet: FOXO4 overexpression abrogates the miR-328-3p-mediated cell injury. THLE-2 cells were co-transfected with pHBV1.3 or empty pcDNA3.1 vector (control), and miR-328-3p mimic or mimic negative control (NC), under LPS (1 μg/mL, 24 h) and HBV stimulation. a Cell proliferation at different time points was examined by MTT assay. b Cell apoptosis was examined by flow cytometry and the apoptosis rate was shown. c Levels of TNF-α, IL-6, IL-8, IL-12, and IL-18 were measured by ELISA. Values are presented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01 vs. LPS + HBV + Vector + mimic NC. # P < 0.05, ## P < 0.01 vs. LPS + HBV + Vector + miR-328-3p mimic

Article Snippet: The HBV replication plasmid pHBV1.3 which contained 1.3 copies of the HBV genome was obtained from BioVector (Beijing, China).

Techniques: Over Expression, Transfection, Plasmid Preparation, Negative Control, MTT Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay