Journal: PLoS ONE

Article Title: TRAIL Enhances Apoptosis of Human Hepatocellular Carcinoma Cells Sensitized by Hepatitis C Virus Infection: Therapeutic Implications

doi: 10.1371/journal.pone.0098171

Figure Lengend Snippet: (A) Western blot analysis of Bax and cleaved caspase-8, caspase-9, and caspase-3 in HCV-infected cells. Huh7.5.1 cells infected with HCVcc in the absence or presence of recombinant TRAIL (100 ng/ml) were used for Western blot analysis with antibodies against the indicated proteins. β-actin was used as an internal loading control. Samples: MC, media control. (B and D) Silencing DR4 and DR5 prevent caspase-9 cleavage and caspase-3/7 activity induced by HCV infection. Huh7.5.1 cells transfected with non-targeting (NT) or gene-specific (ST) siRNA pools targeting DR4 and DR5, respectively, were infected with HCVcc. At 3 days post-infection, the mRNA levels of DR4 and DR5 were analyzed by real-time qRT-PCR (B) (mean±SD; n = 3; *p<0.05, **p<0.01) and whole cell lysates were analyzed by Western blotting with antibodies specific to the indicated proteins (C). (D) The activity of caspase-3/7 was measured according to manufacturer's instructions (mean±SD; n = 3; *p<0.005).

Article Snippet: Primary antibodies used in this study include the following: rabbit monoclonal anti-cleaved PARP (Cell signaling Technology); mouse monoclonal anti-DR4 (Abcam); rabbit monoclonal anti-DR5 (Cell signaling Technology); rabbit monoclonal anti-cleaved caspase-3 (Cell signaling Technology); rabbit monoclonal anti-cleaved caspase-8 (Cell signaling Technology); rabbit monoclonal anti-cleaved caspase-9 (Cell signaling Technology); rabbit monoclonal anti-cleaved Bid (Invitrogen); mouse monoclonal anti-HCV core (Thermo Fisher Scientific); mouse monoclonal anti-α-tubulin (Sigma); mouse monoclonal anti-β-actin (Sigma).

Techniques: Western Blot, Infection, Recombinant, Activity Assay, Transfection, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: TRAIL Enhances Apoptosis of Human Hepatocellular Carcinoma Cells Sensitized by Hepatitis C Virus Infection: Therapeutic Implications

doi: 10.1371/journal.pone.0098171

Figure Lengend Snippet: (A) Western blot analysis of cleaved PARP in HCV-infected cells. Whole cell lysates extracted from HCV-infected Huh7.5.1 cells grown in the absence or presence of recombinant TRAIL (100 ng/ml) were analyzed by Western blotting with antibodies specific to the indicated proteins. (B) Huh7.5.1 cells transfected with non-targeting (NT) or gene-specific (ST) siRNA pools targeting DR4 and DR5, respectively, were infected with HCVcc. At 3 days post-infection, whole cell lysates were analyzed by Western blotting with antibodies specific to the indicated proteins. (C) HCV-infected Huh7.5.1 cells stimulated with recombinant TRAIL (100 ng/ml) were treated with Z-VAD-FMK (pan inhibitor; 40 µM), Z-IETD-FMK (caspase-8 inhibitor; 40 µM), and Z-LEHD-FMK (caspase-9 inhibitor; 40 µM), respectively, for 72 h before harvest. The expression level of PARP was analyzed by Western blotting with anti-cleaved PARP antibody. (D) Western blot analysis showing dose-dependent effects of recombinant TRAIL on PARP cleavage in HCV-infected cells. HCV-infected Huh7.5.1 cells were treated with recombinant TRAIL for 72 h at the indicated dose points. Whole cell lysates were analyzed by Western blotting with anti-cleaved PARP antibody. Samples: MC, media control. (A-D) β-actin was used as an internal loading control.

Article Snippet: Primary antibodies used in this study include the following: rabbit monoclonal anti-cleaved PARP (Cell signaling Technology); mouse monoclonal anti-DR4 (Abcam); rabbit monoclonal anti-DR5 (Cell signaling Technology); rabbit monoclonal anti-cleaved caspase-3 (Cell signaling Technology); rabbit monoclonal anti-cleaved caspase-8 (Cell signaling Technology); rabbit monoclonal anti-cleaved caspase-9 (Cell signaling Technology); rabbit monoclonal anti-cleaved Bid (Invitrogen); mouse monoclonal anti-HCV core (Thermo Fisher Scientific); mouse monoclonal anti-α-tubulin (Sigma); mouse monoclonal anti-β-actin (Sigma).

Techniques: Western Blot, Infection, Recombinant, Transfection, Expressing

Journal: PLoS ONE

Article Title: TRAIL Enhances Apoptosis of Human Hepatocellular Carcinoma Cells Sensitized by Hepatitis C Virus Infection: Therapeutic Implications

doi: 10.1371/journal.pone.0098171

Figure Lengend Snippet: Huh7.5.1 cells were infected with HCVcc in the absence or presence of caspase-8 inhibitor (20 µM) for 72 h before harvest. The expression levels of truncated Bid (t-Bid) and cleaved caspase-9 were analyzed by Western blotting with antibodies specific to t-Bid and cleaved caspase-9. α-tubulin was used as an internal loading control. The relative intensity of t-Bid and cleaved caspase-9 normalized to α-tubulin was analyzed by ImageJ. Samples: MC, media control.

Article Snippet: Primary antibodies used in this study include the following: rabbit monoclonal anti-cleaved PARP (Cell signaling Technology); mouse monoclonal anti-DR4 (Abcam); rabbit monoclonal anti-DR5 (Cell signaling Technology); rabbit monoclonal anti-cleaved caspase-3 (Cell signaling Technology); rabbit monoclonal anti-cleaved caspase-8 (Cell signaling Technology); rabbit monoclonal anti-cleaved caspase-9 (Cell signaling Technology); rabbit monoclonal anti-cleaved Bid (Invitrogen); mouse monoclonal anti-HCV core (Thermo Fisher Scientific); mouse monoclonal anti-α-tubulin (Sigma); mouse monoclonal anti-β-actin (Sigma).

Techniques: Infection, Expressing, Western Blot

Journal: PLoS ONE

Article Title: Depletion of the Adaptor Protein NCK Increases UV-Induced p53 Phosphorylation and Promotes Apoptosis

doi: 10.1371/journal.pone.0076204

Figure Lengend Snippet: (A) HeLa cells transfected with control or NCK1 and NCK2 siRNA were treated with 50 J/m 2 UV and allowed to recover for 2 hr before being fixed and stained with the indicated antibody and DRAQ5. Scale bars are 20 µm. All images are confocal sections. section n = 3. (B) Cells were treated as in A, and equal amounts of lysates were immunoblotted for cleaved CASP3, total CASP3, cleaved PARP, total PARP, and NCK. RAN was used as a loading control. n = 4. (C) HeLa cells transfected with control or NCK1 and NCK2 siRNA were treated with 50 J/m 2 UV and allowed to recover for the indicated amount of time before cell viability was assayed. Percent cell viability for each siRNA at time 0 hr (no treat), is defined as 100%. n = 8; error bars represent SE; (*) P < 0.05, (**) P < 0.01, (#) P < 0.001, (# #) P < 0.0001.

Article Snippet: Primary antibodies used were: rabbit anti-NCK (a gift from Tony Pawson (Univ. of Toronto, Canada)), rabbit anti-cleaved CASP3 (Cell Signaling), rabbit anti-CASP3 (Cell Signaling), rabbit anti-PARP (detects total and cleaved forms) (Cell Signaling), rabbit anti-RAN (described previously [ ]), rabbit anti-p53-pS15 (Cell Signaling), rabbit anti-CHK2-pT68 (Cell Signaling), sheep anti-p53 (Millipore), rabbit anti-NCK2 (a gift from Louise Larose (McGill Univ., Canada)), rabbit anti-CHK2 (Santa Cruz), rabbit anti-γH2AX (pS139) (Novus Biologicals).

Techniques: Transfection, Staining

Journal: PLoS ONE

Article Title: Depletion of the Adaptor Protein NCK Increases UV-Induced p53 Phosphorylation and Promotes Apoptosis

doi: 10.1371/journal.pone.0076204

Figure Lengend Snippet: (A) HeLa cells transfected with control or SOCS7 siRNA were treated with 50 J/m 2 UV and allowed to recover for 1 hr before being fixed and stained with the indicated antibodies and DRAQ5. Scale bars are 20 µm. All images are confocal sections. Bar graphs below are ratio of nuclear to cytoplasmic fluorescence of NCK with siControl, no treat, defined as 1. n = 43-67; error bars represent SE; (**) P < 0.0001. (B) HeLa cells were treated as in A, and equal amounts of lysates were immunoblotted for cleaved CASP3, total CASP3, cleaved PARP, total PARP, and NCK. RAN was used as a loading control. n = 3. (C) RT-PCR of SOCS7 mRNA from HeLa cells transfected with control or SOCS7 siRNA. GAPDH was used as an internal control. RT = reverse transcriptase. n = 3. (D) HeLa cells transfected with control, NCK1 and NCK2, or SOCS7 siRNA were treated with 50 J/m 2 UV and allowed to recover for 2 hr before lysates were prepared. Equal amounts of lysates were immunoblotted for p53-pS15 (phospho-specific), CHK2-pT68 (phospho-specific), and NCK. RAN was used as a loading control. Band intensities were measured with ImageJ. Bar graph is ratio of p53-pS15 band intensity to RAN band intensity with siControl, no treat (NT), defined as 1. n = 4; error bars represent SE; (*) P < 0.005. (E) RT-PCR of p53 mRNA from HeLa cells transfected with control, NCK1 and NCK2, or SOCS7 siRNA. GAPDH was used as an internal control. Bar graph is ratio of p53 mRNA to GAPDH mRNA with siControl ratio defined as 1. n = 4; error bars represent SE. (F) 293T cells transfected with control, NCK1 and NCK2, or SOCS7 siRNA were treated with 50 J/m 2 UV and allowed to recover for 2 hr before lysates were prepared. Equal amounts of lysates were immunoblotted for p53-pS15, p53 (total protein), and NCK. RAN was used as a loading control. n = 3. (G) RT-PCR of SOCS7 mRNA from 293T cells transfected with control or SOCS7 siRNA. GAPDH was used as an internal control. n = 3.

Article Snippet: Primary antibodies used were: rabbit anti-NCK (a gift from Tony Pawson (Univ. of Toronto, Canada)), rabbit anti-cleaved CASP3 (Cell Signaling), rabbit anti-CASP3 (Cell Signaling), rabbit anti-PARP (detects total and cleaved forms) (Cell Signaling), rabbit anti-RAN (described previously [ ]), rabbit anti-p53-pS15 (Cell Signaling), rabbit anti-CHK2-pT68 (Cell Signaling), sheep anti-p53 (Millipore), rabbit anti-NCK2 (a gift from Louise Larose (McGill Univ., Canada)), rabbit anti-CHK2 (Santa Cruz), rabbit anti-γH2AX (pS139) (Novus Biologicals).

Techniques: Transfection, Staining, Fluorescence, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Depletion of the Adaptor Protein NCK Increases UV-Induced p53 Phosphorylation and Promotes Apoptosis

doi: 10.1371/journal.pone.0076204

Figure Lengend Snippet: (A) HeLa cells transfected with GFP-NCK1 or GFP-NCK2 were treated with 50 J/m 2 UV and allowed to recover for 2 hr before being fixed and stained with indicated antibodies and DRAQ5. Scale bars are 20 µm. All images are confocal sections. Bar graphs below each panel are ratio of nuclear to cytoplasmic fluorescence of GFP-NCK with no treat defined as 1. n = 14-28; error bars represent SE; (*) P < 0.001, (**) P < 0.0001. (B) HeLa cells transfected with control, NCK1, or NCK2 siRNA were treated with 50 J/m 2 UV and allowed to recover for 2 hr before lysates were prepared. Equal amounts of lysates were immunoblotted for p53-pS15 (phospho-specific) and NCK. RAN was used as a loading control. n = 3. (C) RT-PCR of p53 mRNA from HeLa cells transfected with control, NCK1, or NCK2 siRNA. GAPDH was used as an internal control. RT = reverse transcriptase. n = 4. (D) HeLa cells transfected with control, NCK1, or NCK2 siRNA were treated with 50 J/m 2 UV and allowed to recover for 2 hr before lysates were prepared. Equal amounts of lysates were immunoblotted for cleaved CASP3, total CASP3, cleaved PARP, total PARP, and NCK. RAN was used as a loading control. n = 3. (E) RT-PCR of NCK1 and NCK2 mRNA from HeLa cells transfected with control, NCK1, or NCK2 siRNA. GAPDH was used as an internal control. n = 3. (F) Cell lysates from HeLa cells transfected with control, NCK1, or NCK2 siRNA were immunoprecipitated with a NCK antibody, which detects NCK 1 and NCK2, or IgG control, and immunoblotted for NCK or NCK2. n = 4.

Article Snippet: Primary antibodies used were: rabbit anti-NCK (a gift from Tony Pawson (Univ. of Toronto, Canada)), rabbit anti-cleaved CASP3 (Cell Signaling), rabbit anti-CASP3 (Cell Signaling), rabbit anti-PARP (detects total and cleaved forms) (Cell Signaling), rabbit anti-RAN (described previously [ ]), rabbit anti-p53-pS15 (Cell Signaling), rabbit anti-CHK2-pT68 (Cell Signaling), sheep anti-p53 (Millipore), rabbit anti-NCK2 (a gift from Louise Larose (McGill Univ., Canada)), rabbit anti-CHK2 (Santa Cruz), rabbit anti-γH2AX (pS139) (Novus Biologicals).

Techniques: Transfection, Staining, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation

Journal: Experimental and Therapeutic Medicine

Article Title: LncRNA NEAT1/microRNA-124 regulates cell viability, inflammation and fibrosis in high-glucose-treated mesangial cells

doi: 10.3892/etm.2022.11434

Figure Lengend Snippet: Antibodies applied for western blotting in the present study.

Article Snippet: Rabbit monoclonal to Cleaved , Cell Signaling , 1:1,000 , ab9664.

Techniques: Western Blot