Journal: PLoS ONE

Article Title: Characterization of Rhodamine-123 as a Tracer Dye for Use In In vitro Drug Transport Assays

doi: 10.1371/journal.pone.0033253

Figure Lengend Snippet: (A) Critical micelle concentration of R123 was determined by measuring quantum yield (λex = 505 nm, λem = 5251nm) of 0–0.5 µM R123 in 1% (v/v)MeOH:HBSS. (B) MDCKII-ABCB1 cells were incubated with 0–20 µM R123 for 10 minutes and intracellular R123 concentration determined against a standard curve following cell lysis with Triton ×100. (C) MDCKII or Huh7 cells were incubated with 10 µM R123 for 10 minutes and R123 concentration in soluble and insoluble cellular fractions determined against R123 standard curves. (D) Albumin binding was determined through the measurement of 0.1 µM R123 fluorescence following the addition of increasing quantities of 0–15 µM albumin. (E) S9 metabolic fractions were extracted from human liver samples and MDCKII cells and used at a final concentration of and respectively. The rate of conversion of R123 to R110 was determined following addition of 0.1 mg/ml or 1 mg/ml S9 fraction from MDCKII cells or human liver, respectively. Rate of conversion was determined for the indicated range of R123, and fitted using an allosteric sigmoidal model of enzyme kinetics. Data points are the average of three separate repeats ± S.D; where no error bars are observed, they are contained within the limits of the data point.

Article Snippet: The human hepatocellular carcinoma (Huh7) cell line is a differentiated, immortalised cell line commonly used as an in vitro hepatocyte model , , and was a kind gift of GlaxoSmithKline.

Techniques: Concentration Assay, Incubation, Lysis, Binding Assay, Fluorescence

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