Journal: Frontiers in Genetics

Article Title: An in vitro Study on the Role of Hepatitis B Virus X Protein C-Terminal Truncation in Liver Disease Development

doi: 10.3389/fgene.2021.633341

Figure Lengend Snippet: Cell growth and proliferation. (A) MTT assay. 10,000 cells/well were seeded in a 96-well plate and transfected with empty pcDNA3, pcDNA3-HBx, and pcDNA3-HBxΔ127. Cells were subjected to MTT assay 4 days post-transfection. Experiments were performed in dark and optical density (OD) at 570 nm taken using a microplate reader. Relative OD values were calculated using values obtained for empty-pcDNA3 transfected wells as reference. Data shown as mean ± SD of three independent experiments performed in quadruplicate. (B) Cell cycle analysis. Huh7 cells were seeded in a 6-well plate, and after the cells grew 70% confluent, the cells were transfected with empty pcDNA3, pcDNA3-HBx, and pcDNA3-HBxΔ127. Forty-eight hours post-transfection, cells were fixed in 70% ethanol, stained with propidium iodide, and analyzed using a flow cytometer. Histograms represent one of the three independent experiments. Bar graphs show percentage of cells in G0/G1, and S + G2/M phases of cell cycle for empty-pcDNA3-transfected cells (control), and HBx- and HBxΔ127-transfected cells. Bar graphs represent mean ± SD of three independent experiments performed in duplicate. (C) Cell growth assay. Cells (100,000/well) were seeded in a 12-well plate, and at 70% confluency, the cells were transfected with empty pcDNA3, pcDNA3-HBx, and pcDNA3-HBxΔ127. Cells were maintained in culture conditions for 36 h, then harvested and replated (8,000 cells/well) in a 12-well plate. Cells were allowed to grow for five more days in the new culture plate and then stained with crystal violet. Bar graph shows quantification of crystal violet in the stained cells. Crystal violet retrieved in 0.1% SDS solution was quantified by taking optical density (OD) at 570 nm using a spectrophotometer. Data are shown as mean ± SD of three independent experiments performed in duplicate. For (A–C) data are analyzed by paired two-tailed Student’s t -test; * P < 0.05 compared with the control.

Article Snippet: Human hepatoma cell line Huh7 was cultured in Dulbecco’s Modified Eagle’s Medium, 10% fetal bovine serum, and penicillin–streptomycin (100 μg/ml each) (Genetix Biotech) in an incubator at 37°C in a humid atmosphere with 5% CO 2 .

Techniques: MTT Assay, Transfection, Cell Cycle Assay, Staining, Flow Cytometry, Control, Growth Assay, Spectrophotometry, Two Tailed Test

Journal: Frontiers in Genetics

Article Title: An in vitro Study on the Role of Hepatitis B Virus X Protein C-Terminal Truncation in Liver Disease Development

doi: 10.3389/fgene.2021.633341

Figure Lengend Snippet: (A) Mitochondrial depolarization studied by tetramethylrhodamine ethyl ester (TMRE) staining. 10,000 cells/well were seeded in a 96-well plate and transfected with empty pcDNA3, pcDNA3-HBx, and pcDNA3-HBxΔ127. Forty-eight hours after transfection, cells were treated with TMRE and fluorescence measured by a microplate reader (Varioskan Flash Multimode Reader; Thermo Scientific) at excitation wavelength 549 nm and emission wavelength 575 nm. (B) Reactive oxygen species (ROS) production studied by dihydroethidium (DHE) staining. Huh7 cells cultured in a 12-well plate and transfected with empty pcDNA3, pcDNA3-HBx, and pcDNA3-HBxΔ127. Intracellular ROS formation was checked 48 h after transfection by treatment with DHE stain. Cells take DHE stain in proportion to their intracellular ROS level. As a positive control, dead cells prepared by prolonged media starvation were used. All the dead cells were 100% DHE positive. Empty-pcDNA3-transfected control cells used as negative control. Almost nil staining was observed for negative control. Significant increase in percentage of DHE-positive cells was observed in cell cultures transfected with HB×Δ127 as compared with wtHBx. Images were taken by fluorescent inverted microscope, 10× objective (Nikon, Tokyo, Japan). Scale bar, 100 μm. Identical acquisition parameters were used while taking these images. Images shown here represent one of the three independent experiments performed in triplicate.

Article Snippet: Human hepatoma cell line Huh7 was cultured in Dulbecco’s Modified Eagle’s Medium, 10% fetal bovine serum, and penicillin–streptomycin (100 μg/ml each) (Genetix Biotech) in an incubator at 37°C in a humid atmosphere with 5% CO 2 .

Techniques: Staining, Transfection, Fluorescence, Cell Culture, Positive Control, Control, Negative Control, Inverted Microscopy

Journal: Frontiers in Genetics

Article Title: An in vitro Study on the Role of Hepatitis B Virus X Protein C-Terminal Truncation in Liver Disease Development

doi: 10.3389/fgene.2021.633341

Figure Lengend Snippet: Huh7 cells cultured in 24-well plate and experiments performed at 70% confluency. Observations done after treating cells with DAPI antifade mountant 24 h post-transfection. (A1,B1,C1) Images showing DAPI-stained nuclei. (A2,B2,C2) Images showing overlap of (A1,B1,C1 , respectively, with images of the same view field captured to observe GFP-HBx expression. (A1,A2) Loose aggregate of cells expressing GFP-HBx visible in cell cultures transfected with pEGFP-C3-HBx. (B1,B2,C1,C2) Tumor initiation clumps (TIC) visible in cell cultures transfected with pEGFP-C3-HBxΔ127. All observations done under inverted fluorescence microscope, 40× objective. Scale bar, 30 μm. Identical acquisition parameters were used while taking images. Refer to to see normal GFP expression and morphology of huh7 nuclei.

Article Snippet: Human hepatoma cell line Huh7 was cultured in Dulbecco’s Modified Eagle’s Medium, 10% fetal bovine serum, and penicillin–streptomycin (100 μg/ml each) (Genetix Biotech) in an incubator at 37°C in a humid atmosphere with 5% CO 2 .

Techniques: Cell Culture, Transfection, Staining, Expressing, Fluorescence, Microscopy

Journal: Frontiers in Genetics

Article Title: An in vitro Study on the Role of Hepatitis B Virus X Protein C-Terminal Truncation in Liver Disease Development

doi: 10.3389/fgene.2021.633341

Figure Lengend Snippet: (A1–A5) Huh7 cells transfected with pEGFP-C3-HBx, and effect of post-transfection incubation time on expression pattern of GFP-HBx observed. Images captured 18–24 h (A1,A2) , 36–48 h (A3) , 48–72 h (A4) , 72–96 h, and onward (A5) post-transfection. (B1,B2) Expression of GFP-HBxΔ127 observed in cells transfected with pEGFP-C3-HBxΔ127, 18 h post-transfection. Expression of single granular body of GFP-HBxΔ127 at one pole of the nucleus observed as early as 18 h after cells were transfected with pEGFP-C3-HBxΔ127. Observations were done using an inverted fluorescence microscope, 40× objective. Scale bar, 30 μm. Identical acquisition parameters used while taking images.

Article Snippet: Human hepatoma cell line Huh7 was cultured in Dulbecco’s Modified Eagle’s Medium, 10% fetal bovine serum, and penicillin–streptomycin (100 μg/ml each) (Genetix Biotech) in an incubator at 37°C in a humid atmosphere with 5% CO 2 .

Techniques: Transfection, Incubation, Expressing, Fluorescence, Microscopy

Journal: Cancer Cell International

Article Title: Hsa-miR-423-5p selectively loaded in hypoxic exosomes reduces the sensitivity of normoxic hepatocellular carcinoma to sorafenib via autophagy

doi: 10.1186/s12935-025-04055-8

Figure Lengend Snippet: HExs reduced the sensitivity of normoxic HCC cells to sorafenib via autophagy. A & B . Following pretreatment of the cells with exosomes, Annexin V-FITC/PI flow cytometry was used to determine the extent of sorafenib-induced apoptosis. C . The IC50 of sorafenib in Huh7 and 97H cells was detected using a CCK-8 assay. D . Huh7 and 97H cells were preincubated with or without HExs and subsequently treated with sorafenib (12, 24, and 48 h). The final assessment of cell viability was performed using a CCK-8 assay. E & F . After autophagy, sorafenib-induced apoptosis was detected via Annexin V-FITC/PI flow cytometry. G & H . Western blot analysis was used to detect the expression levels of autophagy markers in Huh7 and 97H cells that had been preincubated with or without hypoxic exosomes and subsequently treated with sorafenib (* p < 0.05, ** p < 0.01)

Article Snippet: The human hepatoma cell line Huh7, human hepatoma cell line MHCC-97H (97H) and human renal epithelial cell line 293 T were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Flow Cytometry, CCK-8 Assay, Western Blot, Expressing

Journal: Cancer Cell International

Article Title: Hsa-miR-423-5p selectively loaded in hypoxic exosomes reduces the sensitivity of normoxic hepatocellular carcinoma to sorafenib via autophagy

doi: 10.1186/s12935-025-04055-8

Figure Lengend Snippet: Hsa-miR-423-5p was selectively loaded by HExs. A & B . Heatmaps and volcano plots of differentially expressed miRNAs between NExs and HExs secreted by Huh7 or 97H cells. C . Venn diagram of upregulated miRNAs in HExs secreted by Huh7 and 97H cells. D . The expression of the target miRNAs (hsa-miR-29a-3p and hsa-miR-1273f) in exosomes and cells was verified by RT‒qPCR. E & F . Heatmaps and volcano plots of differentially expressed miRNAs in Huh7 and 97H cells (hypoxic vs. normoxic) and exosomes (HExs vs. NExs). G . Venn diagram of miRNAs whose expression levels were not significantly changed in hypoxic HCC cells, downregulated in hypoxic HCC cells, or upregulated in HExs. H . The expression levels of hsa-miR-423-5p in exosomes and cells were verified by RT‒qPCR (N.S. p ≥ 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: The human hepatoma cell line Huh7, human hepatoma cell line MHCC-97H (97H) and human renal epithelial cell line 293 T were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Expressing

Journal: Cancer Cell International

Article Title: Hsa-miR-423-5p selectively loaded in hypoxic exosomes reduces the sensitivity of normoxic hepatocellular carcinoma to sorafenib via autophagy

doi: 10.1186/s12935-025-04055-8

Figure Lengend Snippet: HNRNPA1 mediated the selective loading of hsa-miR-423-5p in HExs. A . The catRAPID website predicted RNA-binding proteins that could interact with hsa-miR-423-5p. B . HNRNPA1 expression was detected by Western blotting in Huh7 and 97H cells cultured under normoxic or hypoxic conditions. C . RNA pull-down experiments were performed using the miR-423-5p probe to isolate RNA-binding proteins, and the expression level of HNRNPA1 was subsequently assessed by Western blot analysis. D . RIP experiments were performed using an HNRNPA1-specific antibody to pull down miRNAs, and the expression level of hsa-miR-423-5p was subsequently measured by RT‒qPCR. E . Huh7 and 97H cells were infected with a specific knockdown lentivirus (sh-HNRNPA1) or a control lentivirus (sh-NC). The knockdown efficiency of sh-HNRNPA1 on HNRNPA1 was assessed by Western blotting. F . Cells were collected after infection with a lentivirus, along with their secreted exosomes. The expression levels of hsa-miR-423-5p in both cells and exosomes were measured by qRT-PCR (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: The human hepatoma cell line Huh7, human hepatoma cell line MHCC-97H (97H) and human renal epithelial cell line 293 T were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: RNA Binding Assay, Expressing, Western Blot, Cell Culture, Infection, Knockdown, Control, Quantitative RT-PCR

Journal: Cancer Cell International

Article Title: Hsa-miR-423-5p selectively loaded in hypoxic exosomes reduces the sensitivity of normoxic hepatocellular carcinoma to sorafenib via autophagy

doi: 10.1186/s12935-025-04055-8

Figure Lengend Snippet: Hsa-miR-423-5p promoted autophagy in normoxic HCC cells by targeting TAB2, thereby reducing the sensitivity of normoxic HCC cells to sorafenib . A . Huh7 and 97H cells were transfected with miR-423-5p mimics or mimics-NC, and the expression level of hsa-miR-423-5p in the cells was detected by RT‒qPCR. B . Expression levels of the autophagy markers LC3B and SQSTM1 in cells were detected by Western blotting. C . Huh7 and 97H cells were transfected with or without the miR-423-5p mimic, followed by sorafenib treatment, and apoptosis was detected via Annexin V-FITC/PI flow cytometry. D . Wild-type and mutated binding sites between hsa-miR-423-5p and TAB2. Relative luciferase intensity of cells cotransfected with miR-423-5p mimics or mimics-NC and TAB2-WT or TAB2-MUT. E . Expression level of TAB2 in Huh7 and 97H cells transfected with miR-423-5p mimics or mimics-NC (N.S. p ≥ 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: The human hepatoma cell line Huh7, human hepatoma cell line MHCC-97H (97H) and human renal epithelial cell line 293 T were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Transfection, Expressing, Western Blot, Flow Cytometry, Binding Assay, Luciferase