Journal: Virology Journal

Article Title: Permissiveness of human hepatoma cell lines for HCV infection

doi: 10.1186/1743-422X-9-30

Figure Lengend Snippet: Expression of HCV entry factors in hepatoma cells lines . ( A ) Total RNA was extracted from Huh7, PLC, Hep3B and HepG2-CD81 cells and mRNA copies were determined by RTqPCR using standard curves comprised of DNA plasmids expressing the coding sequence of the gene of interest. Absolute quantities were normalized to GAPDH and data are displayed as mRNA copies/μg total cellular RNA (means ± SD). ( B-C ) Cell surface expression of HCV entry factors on Huh7, PLC, Hep3B and HepG2-CD81 cells was determined by flow cytometric analysis or IF. ( B ) For flow cytometric analysis, cells were trypsinized and stained with mouse anti-CD81 or anti-SR-BI monoclonal antibodies and an anti-mouse secondary antibody conjugated with PE. Shaded regions represent cells stained with a monoclonal mouse IgG control primary antibody and an anti-mouse PE-conjugated secondary antibody. ( C ) For IF analysis, fixed cells were stained with antibodies specific for CLDN1 or OCLN and counterstained with a species specific Alexa-555-conjugated secondary antibody. Indicated protein is red (Alexa 555) and nuclei are blue (Hoechst). Magnification × 630; scale bar = 20 μm.

Article Snippet: Cells were stained with a 1:750 dilution of a mouse anti-human CLDN1 (Abnova, Taipei, Taiwan) or mouse anti-human OCLN (Zymed, San Francisco, CA) primary antibody overnight at 4°C, followed by incubation with a 1:750 dilution of an anti-mouse Alexa-555 conjugated secondary antibody (Molecular Probes) for 1 h at room temperature.

Techniques: Expressing, Sequencing, Staining, Control

Journal: Virology Journal

Article Title: Permissiveness of human hepatoma cell lines for HCV infection

doi: 10.1186/1743-422X-9-30

Figure Lengend Snippet: HCVpp and HCVcc infection in PLC, Hep3B and HepG2-CD81 cells exogenously over expressing HCV entry factors . Huh7, PLC, Hep3B and HepG2-CD81 cells transiently transfected with a vector control plasmid or expression plasmids expressing CD81, SR-BI, CLDN1 and/or OCLN were infected with ( A-C ) equal amounts of JFHpp, H77pp or VSVGpp or ( D-F ) HCVcc at an MOI of 2.0 FFU/cell. HCVpp entry (following background and mock-control signal subtraction) is expressed as relative light units (RLU) ± SD for triplicate samples determined 72 h p.i. Intracellular HCVcc RNA was quantified by RTqPCR, normalized to GAPDH and is displayed as mean HCV RNA copies/μg total cellular RNA ± SD for triplicate samples determined 72 h p.i.

Article Snippet: Cells were stained with a 1:750 dilution of a mouse anti-human CLDN1 (Abnova, Taipei, Taiwan) or mouse anti-human OCLN (Zymed, San Francisco, CA) primary antibody overnight at 4°C, followed by incubation with a 1:750 dilution of an anti-mouse Alexa-555 conjugated secondary antibody (Molecular Probes) for 1 h at room temperature.

Techniques: Infection, Expressing, Transfection, Plasmid Preparation, Control

Journal: PLoS ONE

Article Title: Stage-Specific Expression of TNFα Regulates Bad/Bid-Mediated Apoptosis and RIP1/ROS-Mediated Secondary Necrosis in Birnavirus-Infected Fish Cells

doi: 10.1371/journal.pone.0016740

Figure Lengend Snippet: ( A ) TNFα protein expression level in IPNV-infected ZF4 cells (MOI = 1) 0, 6, 12, and 24 h p.i. The protein was detected using western blot with a polyclonal antibody specific for TNFα. Lanes 1–4: ZF4 cells were pretreated with 50 µM AG-126 and infected with IPNV for 0 (lane 1), 6 (lane 2), 12 (lane 3), or 24 (lane 4). Lanes 5–7: Untreated ZF4 cells were infected with IPNV for 6 (lane 5), 12 (lane 6), or 24 h (lane 7). The expression of actin was used as an internal control. ( B ) TNFα mRNA expression in IPNV-infected ZF4 cells was quantified using RT-PCR. The ZF4 cells were pre-treated with 50 µM or 100 µM AG-126 for 2 hours, infected with IPNV (MOI = 1), and incubated for 0, 3, 6, 12, or 24 h. The expression of ef1a (elongation factor 1-alpha) was used as an internal control. ( C ) The tnfa expression was inhibited by TNFα-specific siRNA in IPNV-infected cells. TNFα expression was efficiently inhibited by TNFα-specific siRNA after IPNV infection. Sample 1: ZF4 cells infected by IPNV. Sample 2: ZF4 cells pretreated with scrambled siRNA and then infected by IPNV. Sample 3: ZF4 cells pretreated with TNFα-specific siRNA and then infected by IPNV. The quantification of gene expression in normal versus siRNA-treated cells was calculated relative to ef1a . ( D ) Detection of TNFα in untreated or TNFα-specific siRNA-treated ZF4 cells after IPNV infection by western blotting. Lane 1: untreated ZF4 cells; lane 2: ZF4 cells treated with control siRNA; lane 3: ZF4 cells treated with TNFα-specific siRNA. The expression of actin was used as an internal control. ( E ) Cell viability of IPNV-infected ZF4 cells pre-treated with TNFα-specific siRNA or AG-126 at 0, 6, 12, 24, 36 and 48 h p.i. The viability of each sample was determined in three individual experiments. Data shown are the mean ± SD. Student's t tests indicate significant differences compared to IPNV infection only or untreated control: *, p <0.05; **, p <0.01.

Article Snippet: Alternatively, the blots were incubated with a 1∶3000 dilution of a polyclonal anti-TNFα antibody (AnaSpec, USA) and a 1∶50,000 dilution of a peroxidase-conjugated goat anti-rabbit antibody (Sigma Aldrich).

Techniques: Expressing, Infection, Western Blot, Control, Reverse Transcription Polymerase Chain Reaction, Incubation, Gene Expression

Journal: PLoS ONE

Article Title: Stage-Specific Expression of TNFα Regulates Bad/Bid-Mediated Apoptosis and RIP1/ROS-Mediated Secondary Necrosis in Birnavirus-Infected Fish Cells

doi: 10.1371/journal.pone.0016740

Figure Lengend Snippet: ( A ) The expression level of the pro-apoptotic proteins Bad and Bid in IPNV-infected ZF4 cells (MOI = 1) at 0, 6, 12, and 24 h p.i. was determined. The proteins were detected using western blot with a polyclonal antibody specific for mouse Bad. Lanes 1–4: ZF4 cells were pretreated with TNFα-specific siRNA and infected with IPNV for 0 (lane 1), 6 (lane 2), 12 (lane 3), or 24 h (lane 4). Lanes 5–7: untreated ZF4 cells were infected with IPNV for 6 (lane 5), 12 (lane 6), or 24 h (lane 7). The expression of actin was used as an internal control. Results are expressed as the ratio of Bad/actin. The mRNA expression of bad ( B ) and bid ( D ) in IPNV-infected ZF4 cells was quantified using quantitative RT-PCR. ZF4 cells were pre-treated with TNFα-specific siRNA or AG-126 and infected with IPNV (MOI = 1) for 0, 6, 12, or 24 h. The expression of ef1a was used as an internal control. Data shown are mean ± SD. Student's t tests indicate significant differences compared to untreated control: **, p <0.01. ( C ) The expression level of the pro-apoptotic proteins Bid and t-Bid in IPNV-infected ZF4 cells (MOI = 1) at 0, 6 and 12 h p.i. was determined. The proteins were detected using western blot with a polyclonal antibody specific for Bid. Lanes 2–3: ZF4 cells were pretreated with TNFα-specific siRNA and infected with IPNV for 6 (lane 2) or 12 h (lane 3). Untreated ZF4 cells were infected with IPNV for 6 (lane 4) or 12 h (lane 5). Untreated ZF4 cells were infected with IPNV for 0 h (Lane 1). The expression of actin was used as an internal control. Results are expressed as the ratio of Bid/actin or t-Bid/actin.

Article Snippet: Alternatively, the blots were incubated with a 1∶3000 dilution of a polyclonal anti-TNFα antibody (AnaSpec, USA) and a 1∶50,000 dilution of a peroxidase-conjugated goat anti-rabbit antibody (Sigma Aldrich).

Techniques: Expressing, Infection, Western Blot, Control, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Stage-Specific Expression of TNFα Regulates Bad/Bid-Mediated Apoptosis and RIP1/ROS-Mediated Secondary Necrosis in Birnavirus-Infected Fish Cells

doi: 10.1371/journal.pone.0016740

Figure Lengend Snippet: ( A ) Detection of annexin V-positive cells following infection with IPNV. ZF4 cells were pre-treated with TNFα-specific siRNA or AG-126, infected with IPNV (MOI = 1), and incubated for 0, 6, 12, 18 and 24 h. Three individual experiments were performed for each sample. ( B – D ) Caspase-9, -8, and -3 activities were analyzed. ZF4 cells were pre-treated with TNFα-specific siRNA or AG-126 for 2 hours, infected with IPNV (MOI = 1), and incubated for 0, 6, 12, or 24 h. Luminogenic substrate assays were performed in triplicate. Data shown are the mean ± SD. Student's t tests indicate significant differences compared to IPNV infection only: *, p <0.05.

Article Snippet: Alternatively, the blots were incubated with a 1∶3000 dilution of a polyclonal anti-TNFα antibody (AnaSpec, USA) and a 1∶50,000 dilution of a peroxidase-conjugated goat anti-rabbit antibody (Sigma Aldrich).

Techniques: Infection, Incubation

Journal: PLoS ONE

Article Title: Stage-Specific Expression of TNFα Regulates Bad/Bid-Mediated Apoptosis and RIP1/ROS-Mediated Secondary Necrosis in Birnavirus-Infected Fish Cells

doi: 10.1371/journal.pone.0016740

Figure Lengend Snippet: ( A ) Detection of ROS production in TNFα-specific siRNA or AG-126 pre-treated cells after IPNV infection at 0, 6, 12, 18 or 24 h p.i. Fluorescence assays were performed in triplicate. Determination of the percentage of PI-positive cells after IPNV infection. ( B ) Detection of RIP1 in untreated or TNFα-specific siRNA-treated ZF4 cells by western blotting. Lane 1: untreated IPNV-infected ZF4 cells; lane 2: IPNV-infected ZF4 cells treated with scrambled siRNA; lane 3: IPNV-infected ZF4 cells treated with RIP1-specific siRNA. The expression of actin was used as an internal control. Detection of annexin V-positive cells following infection with IPNV. ZF4 cells were pre-treated with RIP1-specific siRNA ( C ), Nec-1 ( D ), DPI ( E ) or BHA ( F ), infected with IPNV (MOI = 1), and incubated for 0, 6, 12, 18 and 24 h. Three individual experiments were performed for each sample. Data shown are the mean ± SD. Student's t tests indicate significant differences compared to IPNV infection only: *, p <0.05.

Article Snippet: Alternatively, the blots were incubated with a 1∶3000 dilution of a polyclonal anti-TNFα antibody (AnaSpec, USA) and a 1∶50,000 dilution of a peroxidase-conjugated goat anti-rabbit antibody (Sigma Aldrich).

Techniques: Infection, Fluorescence, Western Blot, Expressing, Control, Incubation

Journal: PLoS ONE

Article Title: Stage-Specific Expression of TNFα Regulates Bad/Bid-Mediated Apoptosis and RIP1/ROS-Mediated Secondary Necrosis in Birnavirus-Infected Fish Cells

doi: 10.1371/journal.pone.0016740

Figure Lengend Snippet: Detection of PI-positive cells following infection with IPNV. ZF4 cells were pre-treated with TNFα-specific siRNA ( A ), AG-126 ( B ), RIP1-specific siRNA ( C ), Nec-1 ( D ), DPI ( E ), BHA ( F ) and z-VAD ( G ) then infected with IPNV (MOI = 5), and incubated for 12, 18 and 24 h. ( H ) Detection of ROS production in RIP1-specific siRNA-, Nec-1- or DPI-pre-treated cells after IPNV infection at 0, 6, 12, 18 or 24 h p.i. Fluorescence assays were performed in triplicate. Data shown are the mean ± SD. Student's t tests indicate significant differences compared to IPNV infection only or untreated control: *, p <0.05.

Article Snippet: Alternatively, the blots were incubated with a 1∶3000 dilution of a polyclonal anti-TNFα antibody (AnaSpec, USA) and a 1∶50,000 dilution of a peroxidase-conjugated goat anti-rabbit antibody (Sigma Aldrich).

Techniques: Infection, Incubation, Fluorescence, Control

Journal: PLoS ONE

Article Title: Stage-Specific Expression of TNFα Regulates Bad/Bid-Mediated Apoptosis and RIP1/ROS-Mediated Secondary Necrosis in Birnavirus-Infected Fish Cells

doi: 10.1371/journal.pone.0016740

Figure Lengend Snippet: A hypothesis of how pro-inflammatory TNFα is up-regulated by IPNV infection and how it regulates the apoptotic and necrotic death pathways. When a cell is infected with IPNV (E1-S), the virus binds to the cellular receptor, penetrates the cell, uncoats (the entry stage), and up-regulates the expression of TNFα (a) during the early replication stage (0–6 h p.i.). This up-regulation of TNFα regulates the next wave of gene expression, including the pro-apoptotic genes bad and bid , during the early-middle stage of replication (6–12 h p.i.; b), at which point the TNFR necrotic signaling complex and reactive oxygen species (ROS) are produced. During this stage, phosphatidylserine (PS) is externalized and endonuclease is released from the mitochondria, resulting in DNA restructuring and cleavage. Furthermore, the cell finally enters the post-apoptotic, necrotic stage during the middle-late stage of replication (12–18 h p.i.; c). During the late replication stage (18–24 h p.i.; d), the cells are broken down. The TNFα-mediated death signal is halted by treatment with a specific inhibitor of TNFα production. (e) TNFα-specific siRNA or AG-126, which block both the pro-apoptotic Bad/Bid-mediated death pathway and the TNFR/ROS-mediated secondary necrotic death pathway. (f) DPI, Nec-1 or RIP-1-specific siRNA, which block the activity of RIP-1 or Nox1 and block the formation of the TNFR necrotic signaling complex. (g) BHA blocks the formation of ROS.

Article Snippet: Alternatively, the blots were incubated with a 1∶3000 dilution of a polyclonal anti-TNFα antibody (AnaSpec, USA) and a 1∶50,000 dilution of a peroxidase-conjugated goat anti-rabbit antibody (Sigma Aldrich).

Techniques: Infection, Virus, Expressing, Gene Expression, Produced, Blocking Assay, Activity Assay

Journal: PLoS ONE

Article Title: Stage-Specific Expression of TNFα Regulates Bad/Bid-Mediated Apoptosis and RIP1/ROS-Mediated Secondary Necrosis in Birnavirus-Infected Fish Cells

doi: 10.1371/journal.pone.0016740

Figure Lengend Snippet: Primer sequences for duplex siRNA that specifically targeted the mRNA encoding TNFα or RIP1 and scrambled siRNA.

Article Snippet: Alternatively, the blots were incubated with a 1∶3000 dilution of a polyclonal anti-TNFα antibody (AnaSpec, USA) and a 1∶50,000 dilution of a peroxidase-conjugated goat anti-rabbit antibody (Sigma Aldrich).

Techniques: