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: Mechanisms Underlying Insulin Deficiency-Induced Acceleration of β-Amyloidosis in a Mouse Model of Alzheimer's Disease

doi: 10.1371/journal.pone.0032792

Figure Lengend Snippet: ( A ) Real-time qPCR revealed no difference in BACE1 mRNA levels between STZ- and vehicle-treated 5XFAD mouse brains ( n = 4–5 mice per group). ( B, E ) Western blot analysis of hemibrain lysates from vehicle- and STZ-treated 5XFAD mice. Immunoreactive bands for phosphorylated eIF2α (p-eIF2α) and total eIF2α ( C ), phosphorylated PERK (p-PERK) ( D ), and the 17-kDa fragment of activated caspase-3 and GGA3 ( F ) were quantified and expressed as the percentage of vehicle-treated 5XFAD levels ( n = 4–7 mice per group). Levels of p-eIF2α, but not those of total eIF2α, were significantly elevated in STZ-treated 5XFAD mice (* p <0.05 vs. vehicle). STZ treatments also dramatically increased p-PERK levels in 5XFAD mice ( p = 0.05), while cleaved caspase-3 and GGA3 levels were indistinguishable between STZ- and vehicle-treated subjects. All data are presented as mean ± SEM.

Article Snippet: After blocking, membranes were probed with anti-insulin (1∶500, sc-9168, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-insulin receptor (1∶2,000, MABS65, Millipore, Billerica, MA, USA), anti-BACE1 (1∶1,000, MAB5308, Millipore), anti-ADAM10 (1∶2,500, 422751, Calbiochem), anti-PS1 (1∶1,000, 529591, Calbiochem), an antibody that recognizes C-terminal epitope in APP (1∶1,000, C1/6.1, kindly provided by Dr. Paul Mathews, Nathan Kline Institute) to detect full-length APP/C-terminal fragments, anti-sAPPα (1∶500, 11088, Immuno-Biological Laboratories, Minneapolis, MN, USA), anti-phospho-eIF2α (Ser51) (1∶1,000, #3398, Cell Signaling Technology, Danvers, MA, USA), anti-eIF2α (1∶2,000, #9722, Cell Signaling Technology), anti-phospho-PERK (Ser713) (1∶500, #649401, BioLegend, San Diego, CA, USA), anti-cleaved caspase-3 (Asp175) (1∶1,000, #9661, Cell Signaling Technology), anti-GGA3 (1∶1,500, #4167, Cell Signaling Technology), anti-neprilysin 1∶2,000, ab951, Abcam, Cambridge, MA, USA), anti-IDE (1∶2,000, PC730, Millipore) or anti-β-actin (1∶15,000, AC-15, Sigma-Aldrich).

Techniques: Western Blot

Journal: FEBS Open Bio

Article Title: Inactivation of the three GGA genes in HeLa cells partially compromises lysosomal enzyme sorting

doi: 10.1002/2211-5463.13040

Figure Lengend Snippet: Sorting of cathepsin D in the absence of GGA proteins. (A) Parental WT, GGA1 −/− , GGA2 −/− , GGA3 −/− , and GGA123 −/− HeLa cell lines were metabolically labeled with [ 35 S]methionine/cysteine and processed as described under ‘ ’. Secreted and intracellular cathepsin D molecules were immunoprecipitated with a polyclonal anti‐cathepsin D antibody, resolved by 10% SDS/PAGE and visualized by autoradiography of the dried gel. A representative autoradiograph is shown. (B) The percentage of cathepsin D in the media (m) was calculated as the ratio of radioactivity in the secreted form divided by the sum of the processed (c) and secreted forms (m). The data shown are the mean ± SD for four independent experiments. (C) Parental WT, GGA123 −/− , and GNPTAB −/− HeLa cell lines were processed for cathepsin D sorting as described in (A). A representative autoradiograph is shown, and data presented are the mean of two independent experiments.

Article Snippet: The following published/validated commercial antibodies against human GGA proteins were used to screen for the GGA knockout cell lines: anti‐GGA1 rabbit monoclonal (Abcam, Cat # ab170956, Cambridge, MA, USA), anti‐GGA2 mouse monoclonal (BD Biosciences, Cat # 612612), and anti‐GGA3 rabbit monoclonal (Cell Signaling, Cat # D66F1, Beverly, MA, USA).

Techniques: Metabolic Labelling, Labeling, Immunoprecipitation, SDS Page, Autoradiography, Radioactivity

Journal: OncoTargets and therapy

Article Title: MicroRNA-27a downregulates the expression of Hsp90 and enhances the radiosensitivity in esophageal squamous cell carcinoma

doi: 10.2147/OTT.S197456

Figure Lengend Snippet: miR-27a downregulated the expression of Hsp90 and radioresistant proteins. Notes: ( A ) Hsp90 mRNA decreased significantly in Eca109 and Eca9706 cells transfected with miR-27a mimics (* P <0.05, ** P <0.01). ( B ) Hsp90 protein was consistent with Hsp90 mRNA. ( C ) The expression level of radioresistant proteins and cleaved PARP in ESCC cells transfected with miR-NC or miR-27a mimics, or in combination with 6 Gy radiation were detected by Western blotting. Abbreviations: ESCC, esophageal squamous cell carcinoma; Hsp90, heat shock protein 90; miR-NC, negative control miRNA.

Article Snippet: Primary antibodies used were anti-Hsp90α rabbit monoclonal antibody, anti-phospho-EGFR Tyr1068 rabbit monoclonal antibody, anti-phospho-c-Raf Ser338 rabbit monoclonal antibody, anti-phospho-Akt Ser473 rabbit monoclonal antibody, anti-cleaved PARP rabbit monoclonal antibody and anti-β-actin rabbit monoclonal antibody (Cell signaling technology, Beverly, MA, USA).

Techniques: Expressing, Transfection, Western Blot, Negative Control

Journal: OncoTargets and therapy

Article Title: MicroRNA-27a downregulates the expression of Hsp90 and enhances the radiosensitivity in esophageal squamous cell carcinoma

doi: 10.2147/OTT.S197456

Figure Lengend Snippet: miR-27a enhanced radiosensitivity of ESCC in xenograft models. Notes: ( A ) Tumor growth curves presented by tumor volume indicated that miR-27a inhibited ESCC xenografts growth after radiation (* P <0.05, ** P <0.001). ( B ) Tumor images of different groups after radiation. ( C and D ) IHC staining was used to detect the expression of Hsp90, p-EGFR and cleaved PARP in the xenograft sections (scale bar =50 μm). * P <0.05, ** P <0.001. Abbreviations: ESCC, esophageal squamous cell carcinoma; NC, negative control; Hsp90, heat shock protein 90.

Article Snippet: Primary antibodies used were anti-Hsp90α rabbit monoclonal antibody, anti-phospho-EGFR Tyr1068 rabbit monoclonal antibody, anti-phospho-c-Raf Ser338 rabbit monoclonal antibody, anti-phospho-Akt Ser473 rabbit monoclonal antibody, anti-cleaved PARP rabbit monoclonal antibody and anti-β-actin rabbit monoclonal antibody (Cell signaling technology, Beverly, MA, USA).

Techniques: Immunohistochemistry, Expressing, Negative Control