hcv-infected min6 cells  (KeyGene Inc)

Journal: PLoS ONE

Article Title: Hepatitis C Virus Induced a Novel Apoptosis-Like Death of Pancreatic Beta Cells through a Caspase 3-Dependent Pathway

doi: 10.1371/journal.pone.0038522

Figure Lengend Snippet: ( A ) The images of MIN6 cells infected with HCV at 24, 48, 72 and 96 hpi. MIN6 cells were mock-infected or infected with 1.0 MOI of the supernatant of HCV-infected Huh7.5.1. Scale bar, 10 µm. ( B ) MIN6 cells were infected with HCV at different MOIs. At indicated time points post-infection, the percentage of viable cells was assessed by MTT assay and plotted versus time. The activity measurements were done in triplicates. Data represent means + SD of three independent experiments (n = 9). * P <0.05, ** P <0.01, compared with respective controls.

Article Snippet: HCV-infected MIN6 cells (1.5×10 6 ) harvested at 48 hpi were subjected to measure the translocation of phosphatidylserine according to the manufacturer’s instructions (Keygene, China).

Techniques: Infection, MTT Assay, Activity Assay

Journal: PLoS ONE

Article Title: Hepatitis C Virus Induced a Novel Apoptosis-Like Death of Pancreatic Beta Cells through a Caspase 3-Dependent Pathway

doi: 10.1371/journal.pone.0038522

Figure Lengend Snippet: ( A, B ) MIN6 cells were mock-infected (a) or infected with 1.0 MOI of the supernatant of HCV-infected Huh7.5.1 (b), ultracentrifugation-purified HCV particles (1.0 MOI) (c), the supernatant of HCV-infected Huh7.5.1 after ultracentrifugation (d), CON1 cell culture medium (e), or the supernatant of HCV-infected Huh7.5.1 after UV radiation treatment (f) at 96 hpi. Light microscopy images (scale bar, 10 µm) ( A ) were obtained and cell viability ( B ) of MIN6 cells was determined by MTT assay. ( C, D ) MIN6 cells were mock-infected or infected with HCV particles (1.0 MOI) with addition of indicated dose of RBV ( C ) or BILN 2061 ( D ) at 96 hpi, cell viability was assessed by MTT assay. ( E ) MIN6 cells were infected with HCV as in . At different time-points, cell number was determined by DNA content staining with the fluorescent dye PI. ( F ) MIN6 cells were treated as in (A). Cell death induced by the indicated treatment was measured by trypan blue exclusion. ( A–F ) All activity measurements were done in triplicates. Data represent means + SD of three independent experiments (n = 9). * P <0.05, ** P <0.01, compared with respective controls.

Article Snippet: HCV-infected MIN6 cells (1.5×10 6 ) harvested at 48 hpi were subjected to measure the translocation of phosphatidylserine according to the manufacturer’s instructions (Keygene, China).

Techniques: Infection, Purification, Cell Culture, Light Microscopy, MTT Assay, Staining, Activity Assay

Journal: PLoS ONE

Article Title: Hepatitis C Virus Induced a Novel Apoptosis-Like Death of Pancreatic Beta Cells through a Caspase 3-Dependent Pathway

doi: 10.1371/journal.pone.0038522

Figure Lengend Snippet: ( A–C ) Confocal image of MIN6 cells stained with Hoechst 33258. Scale bar, 10 µm. Cells were mock infected or infected with 1.0 MOI HCV for 96 h. Cells treated with CHX for 48 h served as an apoptosis positive control. White arrows indicate nuclei ( A ). MIN6 cells were treated as in ( B ). Cells were mock-infected or infected with HCV particles (1.0 MOI) with addition of RBV (50 µM) or BILN 2061 (10 µM) at 96 hpi ( C ). ( D ) Electron microscopic analysis as in (A). Scale bar, 2 µm. All measurements were done in triplicates.

Article Snippet: HCV-infected MIN6 cells (1.5×10 6 ) harvested at 48 hpi were subjected to measure the translocation of phosphatidylserine according to the manufacturer’s instructions (Keygene, China).

Techniques: Staining, Infection, Positive Control

Journal: PLoS ONE

Article Title: Hepatitis C Virus Induced a Novel Apoptosis-Like Death of Pancreatic Beta Cells through a Caspase 3-Dependent Pathway

doi: 10.1371/journal.pone.0038522

Figure Lengend Snippet: MIN6 cells were mock-infected or infected with 1.0 MOI of HCV. ( A ) Confocal image of cells stained with TUNEL at 96 hpi. Scale bar, 10 µm. Quantitative summary of the TUNEL-positive staining is provided on the left. ( B ) Caspase 3 activity levels at 24 and 48 hpi. The caspase 3 activity of the control cells at 0 h after treatment was arbitrarily expressed as 1.0. ( C ) Immunoblot analysis of caspase 3 and PARP at 48, 72, 96 hpi. Cells treated with CHX (50 ng/ml) for 48 h were served as an apoptosis positive control. Immunoblots are representative of at least three independent experiments. Amounts of actin were measured as an internal control to verify equivalent sample loading. ( D ) Diagrams of FITC-Annexin V/PI flow cytometry in a representative experiment at 48 hpi. Numbers in the quadrants indicate the proportions of cells in the corresponding areas (left panel). ( A, B, D ) All measurements were done in triplicates. Data represent means + SD of three independent experiments (n = 9). ** P <0.01, compared with respective controls.

Article Snippet: HCV-infected MIN6 cells (1.5×10 6 ) harvested at 48 hpi were subjected to measure the translocation of phosphatidylserine according to the manufacturer’s instructions (Keygene, China).

Techniques: Infection, Staining, TUNEL Assay, Activity Assay, Western Blot, Positive Control, Flow Cytometry

Journal: PLoS ONE

Article Title: Hepatitis C Virus Induced a Novel Apoptosis-Like Death of Pancreatic Beta Cells through a Caspase 3-Dependent Pathway

doi: 10.1371/journal.pone.0038522

Figure Lengend Snippet: MIN6 cells were mock-infected or infected with 1.0 MOI of HCV. ( A ) Mitochondrial transmembrane potential changes at 48 hpi. Representative images from JC-1 staining are shown. The aggregated form of JC-1, characteristic of mitochondrial integrity (red) and monomeric JC-1 (green) were examined under laser confocal scanning microscope. ( B ) Electron microscopy of mitochondrial morphologies at 96 hpi. Cells treated with CHX (50 ng/ml) for 48 h were served as an apoptosis positive control. Black and white arrows indicate mitochondria and insulin granules, respectively. Scale bar, 1 µm.

Article Snippet: HCV-infected MIN6 cells (1.5×10 6 ) harvested at 48 hpi were subjected to measure the translocation of phosphatidylserine according to the manufacturer’s instructions (Keygene, China).

Techniques: Infection, Staining, Microscopy, Electron Microscopy, Positive Control

Journal: PLoS ONE

Article Title: Hepatitis C Virus Induced a Novel Apoptosis-Like Death of Pancreatic Beta Cells through a Caspase 3-Dependent Pathway

doi: 10.1371/journal.pone.0038522

Figure Lengend Snippet: ( A ) mRNA levels of GRP78 and CHOP. MIN6 cells were mock-infected or infected with 1.0 MOI HCV for 24, 48, 72 h. mRNA was analyzed by real-time RT-PCR. The results were normalized with the values obtained from actin in the same sample. Data are expressed as fold-increase relative to the values observed in mock control. The measurements were done in triplicates. Data represent means + SD of three independent experiments (n = 9). * P <0.05, compared with respective controls. ( B ) Immunoblot analysis of GRP78, CHOP and p-PERK corresponding to (A) probed with the indicated antibodies. Amounts of actin were measured as an internal control to verify equivalent sample loading. Immunoblots are representative of at least three independent experiments.

Article Snippet: HCV-infected MIN6 cells (1.5×10 6 ) harvested at 48 hpi were subjected to measure the translocation of phosphatidylserine according to the manufacturer’s instructions (Keygene, China).

Techniques: Infection, Quantitative RT-PCR, Western Blot

Journal: PLoS ONE

Article Title: Hepatitis C Virus Induced a Novel Apoptosis-Like Death of Pancreatic Beta Cells through a Caspase 3-Dependent Pathway

doi: 10.1371/journal.pone.0038522

Figure Lengend Snippet: ( A ) Kinetics of detection of positive-strand HCV RNA. MIN6 and Huh7.5.1 cells were mock-infected or infected with 1.0 MOI HCV. At different time points, cells were harvested and total RNA was isolated and reverse-transcribed to cDNA. Single-round PCR products (150 bp) were obtained by amplification with POSF/R primers. Actin was measured as an internal control. ( B ) Detection of the synthetic negative-strand RNA as in (A). The sample from HCV-infected Huh7.5.1 cells was used as the positive control. Negative controls included PCR amplification from non-infected cells (−) and water (H 2 O). ( C–D ) MIN6 cells were mock-infected or infected with 1.0 MOI of HCV at 24 hpi. HCV core (red) and NS5A (green) labeled with respective antibody ( C ) or HCV dsRNA labeled with J2 antibody (green) ( D ) were examined by immunofluorescence assay. Blue fluorescence represents DAPI-stained nuclei as observed. ( E ) Immunoprecipitation and blotting of NS5A purified from 96 h-infected MIN6 and Huh7.5.1 cells. Actin from lysis was used as the internal control. All measurements were done in triplicates. Immunoblots are representative of at least three independent experiments.

Article Snippet: HCV-infected MIN6 cells (1.5×10 6 ) harvested at 48 hpi were subjected to measure the translocation of phosphatidylserine according to the manufacturer’s instructions (Keygene, China).

Techniques: Infection, Isolation, Amplification, Positive Control, Labeling, Immunofluorescence, Fluorescence, Staining, Immunoprecipitation, Purification, Lysis, Western Blot