huh7 cells  (atcc)


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    Structured Review

    atcc huh7 cells
    Comparison of overlap between different SARS-CoV-2 genome-wide CRISPR screens A) Upset plot showing overlap of the top 25 hits from this study and previously reported screens B) Enriched GO biological processes for the top 100 resistance hits in Vero E6 and <t>Huh7</t> cells
    Huh7 Cells, supplied by atcc, used in various techniques. Bioz Stars score: 86/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 40 article reviews
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    huh7 cells - by Bioz Stars, 2022-08
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    Images

    1) Product Images from "Systematic genome-scale identification of host factors for SARS-CoV-2 infection across models yields a core single gene dependency; ACE2"

    Article Title: Systematic genome-scale identification of host factors for SARS-CoV-2 infection across models yields a core single gene dependency; ACE2

    Journal: bioRxiv

    doi: 10.1101/2021.06.28.450244

    Comparison of overlap between different SARS-CoV-2 genome-wide CRISPR screens A) Upset plot showing overlap of the top 25 hits from this study and previously reported screens B) Enriched GO biological processes for the top 100 resistance hits in Vero E6 and Huh7 cells
    Figure Legend Snippet: Comparison of overlap between different SARS-CoV-2 genome-wide CRISPR screens A) Upset plot showing overlap of the top 25 hits from this study and previously reported screens B) Enriched GO biological processes for the top 100 resistance hits in Vero E6 and Huh7 cells

    Techniques Used: Genome Wide, CRISPR

    SARS-CoV-2 infection and induced cytopathic effects in human cell types SARS-CoV-2 infectivity and induced cytopathic effects were determined in Vero E6, Calu-3, HEK293 +A+T , Huh7 and Caco-2 cells. Cells were infected with SARS-CoV-2 at MOI of 1 or 10 (Caco-2 cells). At 48 hpi, cells were fixed in 10% NBF and immunostained with anti-SARS N-protein (green), DAPI (blue) and phalloidin (yellow). A) Images were acquired with Phenix Opera system. B) The % cells positive for N-protein staining (green bars, N) was determined by quantifying number of greens cells relative to DAPI stained cells in the same well, while % cell viability was quantified by counting the number of nuclei in the infected cells relative to mock uninfected wells (blue bars, V).
    Figure Legend Snippet: SARS-CoV-2 infection and induced cytopathic effects in human cell types SARS-CoV-2 infectivity and induced cytopathic effects were determined in Vero E6, Calu-3, HEK293 +A+T , Huh7 and Caco-2 cells. Cells were infected with SARS-CoV-2 at MOI of 1 or 10 (Caco-2 cells). At 48 hpi, cells were fixed in 10% NBF and immunostained with anti-SARS N-protein (green), DAPI (blue) and phalloidin (yellow). A) Images were acquired with Phenix Opera system. B) The % cells positive for N-protein staining (green bars, N) was determined by quantifying number of greens cells relative to DAPI stained cells in the same well, while % cell viability was quantified by counting the number of nuclei in the infected cells relative to mock uninfected wells (blue bars, V).

    Techniques Used: Infection, Staining

    2) Product Images from "Mechanisms and Effects on HBV Replication of the Interaction between HBV Core Protein and Cellular Filamin B"

    Article Title: Mechanisms and Effects on HBV Replication of the Interaction between HBV Core Protein and Cellular Filamin B

    Journal: Virologica Sinica

    doi: 10.1007/s12250-018-0023-4

    Filamin B knockdown inhibits the replication of HBV. ( A ) Huh7 and HepG2 cells were transfected with siNC, filamin B-targeting siFLNB-1, siFLNB-2 or siFLNB-3 separately. Western blot analysis of filamin B was used to confirm the silencing efficiency. ( B ) Levels of HBV total RNAs and pgRNA in Huh7 cells were determined by real-time PCR. ( C ) Levels of HBV total RNAs and pgRNA in HepG2 cells were determined by real-time PCR. ( D ) Levels of HBeAg and HBsAg in culture medium supernatant of Huh7 cells were detected with ELISA. ( E ) Levels of HBeAg and HBsAg in culture medium supernatant of HepG2 cells were detected with ELISA. ( F ) HBV DNA was extracted at 96 h post transfection, and then subjected to Southern blotting. Western blot showed that filamin B was successfully knocked down. Co-transfection with pHBV1.3 and siNC was used as a loading control. Ratios were quantified by gray analysis with GeneTools from Syngene software (GeneGnome5). rcDNA, relaxed circular DNA; dsDNA, double stranded DNA; ssDNA, single stranded DNA. All results are shown as mean ± SD. (* P
    Figure Legend Snippet: Filamin B knockdown inhibits the replication of HBV. ( A ) Huh7 and HepG2 cells were transfected with siNC, filamin B-targeting siFLNB-1, siFLNB-2 or siFLNB-3 separately. Western blot analysis of filamin B was used to confirm the silencing efficiency. ( B ) Levels of HBV total RNAs and pgRNA in Huh7 cells were determined by real-time PCR. ( C ) Levels of HBV total RNAs and pgRNA in HepG2 cells were determined by real-time PCR. ( D ) Levels of HBeAg and HBsAg in culture medium supernatant of Huh7 cells were detected with ELISA. ( E ) Levels of HBeAg and HBsAg in culture medium supernatant of HepG2 cells were detected with ELISA. ( F ) HBV DNA was extracted at 96 h post transfection, and then subjected to Southern blotting. Western blot showed that filamin B was successfully knocked down. Co-transfection with pHBV1.3 and siNC was used as a loading control. Ratios were quantified by gray analysis with GeneTools from Syngene software (GeneGnome5). rcDNA, relaxed circular DNA; dsDNA, double stranded DNA; ssDNA, single stranded DNA. All results are shown as mean ± SD. (* P

    Techniques Used: Transfection, Western Blot, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Southern Blot, Cotransfection, Software

    Interaction of filamin B and core protein promotes HBV replication. ( A ) Levels of HBV total RNAs and pgRNA in Huh7 cells were determined by real-time PCR. ( B ) HBV total RNAs and pgRNA levels in HepG2 cells were determined by real-time PCR. ( C ) Levels of HBeAg and HBsAg in culture medium supernatant of Huh7 cells were detected with ELISA. ( D ) Levels of HBeAg and HBsAg in culture medium supernatant of HepG2 cells were detected with ELISA. ( E ) HBV DNA was extracted at 96 h post-transfection and subjected to Southern blotting. Western blot showed that all proteins were overexpressed. Co-transfection with pHBV1.3, GFP and pXJ40-HA was used as a loading control. Ratios were quantified by gray analysis with GeneTools from Syngene software (GeneGnome5). HBV DNA marker bands are shown for relaxed circular DNA (rcDNA), double stranded DNA (dsDNA), and single stranded DNA (ssDNA). All results are shown as mean ± SD. (* P
    Figure Legend Snippet: Interaction of filamin B and core protein promotes HBV replication. ( A ) Levels of HBV total RNAs and pgRNA in Huh7 cells were determined by real-time PCR. ( B ) HBV total RNAs and pgRNA levels in HepG2 cells were determined by real-time PCR. ( C ) Levels of HBeAg and HBsAg in culture medium supernatant of Huh7 cells were detected with ELISA. ( D ) Levels of HBeAg and HBsAg in culture medium supernatant of HepG2 cells were detected with ELISA. ( E ) HBV DNA was extracted at 96 h post-transfection and subjected to Southern blotting. Western blot showed that all proteins were overexpressed. Co-transfection with pHBV1.3, GFP and pXJ40-HA was used as a loading control. Ratios were quantified by gray analysis with GeneTools from Syngene software (GeneGnome5). HBV DNA marker bands are shown for relaxed circular DNA (rcDNA), double stranded DNA (dsDNA), and single stranded DNA (ssDNA). All results are shown as mean ± SD. (* P

    Techniques Used: Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Transfection, Southern Blot, Western Blot, Cotransfection, Software, Marker

    Filamin B interacts with core protein in different cell lines. Co-immunoprecipitation of filamin B and core protein is shown. Either FLNB-GFP plasmid or GFP vector (10 μg) was co-transfected with 10 μg Core-HA plasmid into HEK 293T, Huh7, and HepG2 cells. Filamin B was immunoprecipitated using GFP. Co-immunoprecipitated core protein was detected by immunoblotting with anti-HA antibodies (top). Western blot analysis of the precipitated filamin B and GFP using anti-GFP antibodies is shown (top). Cell lysates before immunoprecipitation were probed with anti-GFP and anti-HA antibodies (bottom).
    Figure Legend Snippet: Filamin B interacts with core protein in different cell lines. Co-immunoprecipitation of filamin B and core protein is shown. Either FLNB-GFP plasmid or GFP vector (10 μg) was co-transfected with 10 μg Core-HA plasmid into HEK 293T, Huh7, and HepG2 cells. Filamin B was immunoprecipitated using GFP. Co-immunoprecipitated core protein was detected by immunoblotting with anti-HA antibodies (top). Western blot analysis of the precipitated filamin B and GFP using anti-GFP antibodies is shown (top). Cell lysates before immunoprecipitation were probed with anti-GFP and anti-HA antibodies (bottom).

    Techniques Used: Immunoprecipitation, Plasmid Preparation, Transfection, Western Blot

    3) Product Images from "Silencing of functional p53 attenuates NAFLD by promoting HMGB1-related autophagy induction"

    Article Title: Silencing of functional p53 attenuates NAFLD by promoting HMGB1-related autophagy induction

    Journal: Hepatology International

    doi: 10.1007/s12072-020-10068-4

    Hepatic HMGB1 translocation induced by p53 silencing. a Immunochemical analysis of HMGB1 expression in livers from p53 +/+ and p53 −/− mice. b Western blotting determination of nuclear, cytoplasmic and total HMGB1 expression in p53 +/+ and p53 −/− mouse livers. c, d Western blotting determination of nuclear, cytoplasmic and total HMGB1 expression in p53-silenced c HepG2 and d Huh7 cells. H3, ACTB and GAPDH were chosen as internal controls for nuclear, cytoplasmic and total protein, respectively. * p
    Figure Legend Snippet: Hepatic HMGB1 translocation induced by p53 silencing. a Immunochemical analysis of HMGB1 expression in livers from p53 +/+ and p53 −/− mice. b Western blotting determination of nuclear, cytoplasmic and total HMGB1 expression in p53 +/+ and p53 −/− mouse livers. c, d Western blotting determination of nuclear, cytoplasmic and total HMGB1 expression in p53-silenced c HepG2 and d Huh7 cells. H3, ACTB and GAPDH were chosen as internal controls for nuclear, cytoplasmic and total protein, respectively. * p

    Techniques Used: Translocation Assay, Expressing, Mouse Assay, Western Blot

    Effect of p53 silencing on autophagy in vitro. a , b Confocal microscopy analysis of autophagosomes in a HepG2 and b Huh7 cells. Cells were first transfected with p53 siRNA for 24 h, followed by pEGFP-LC3 plasmid transfection for another 24 h. DAPI was chosen for nuclear staining. c, d Western blotting analysis of autophagy- and p53-related pathways in p53-silenced c HepG2 and d Huh7 cells. * p
    Figure Legend Snippet: Effect of p53 silencing on autophagy in vitro. a , b Confocal microscopy analysis of autophagosomes in a HepG2 and b Huh7 cells. Cells were first transfected with p53 siRNA for 24 h, followed by pEGFP-LC3 plasmid transfection for another 24 h. DAPI was chosen for nuclear staining. c, d Western blotting analysis of autophagy- and p53-related pathways in p53-silenced c HepG2 and d Huh7 cells. * p

    Techniques Used: In Vitro, Confocal Microscopy, Transfection, Plasmid Preparation, Staining, Western Blot

    p53 silencing ameliorates NAFLD symptoms in vitro. a Intracellular triglyceride levels and b ORO staining of PA-treated primary hepatocytes from p53 +/+ and p53 −/− mice (× 200). c Intracellular triglyceride levels and d ORO staining of p53-silenced HepG2 cells treated with PA (× 200). e Intracellular triglyceride levels and f ORO staining of p53-silenced Huh7 cells treated with different concentrations of PA for 24 h (× 100). * p
    Figure Legend Snippet: p53 silencing ameliorates NAFLD symptoms in vitro. a Intracellular triglyceride levels and b ORO staining of PA-treated primary hepatocytes from p53 +/+ and p53 −/− mice (× 200). c Intracellular triglyceride levels and d ORO staining of p53-silenced HepG2 cells treated with PA (× 200). e Intracellular triglyceride levels and f ORO staining of p53-silenced Huh7 cells treated with different concentrations of PA for 24 h (× 100). * p

    Techniques Used: In Vitro, Staining, Mouse Assay

    4) Product Images from "Medium- and long-chain triglyceride propofol reduces the activity of acetyl-coenzyme A carboxylase in hepatic lipid metabolism in HepG2 and Huh7 cells"

    Article Title: Medium- and long-chain triglyceride propofol reduces the activity of acetyl-coenzyme A carboxylase in hepatic lipid metabolism in HepG2 and Huh7 cells

    Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

    doi: 10.4196/kjpp.2020.24.1.19

    Medium- and long-chain triglyceride propofol inhibited acetyl coenzyme A carboxylase (ACC)1 activity in HepG2 and Huh7 cells. (A) The ACC1 activity in HepG2 cell was detected by ELISA assay. (B) The ACC1 activity in Huh7 cell was measured. * p
    Figure Legend Snippet: Medium- and long-chain triglyceride propofol inhibited acetyl coenzyme A carboxylase (ACC)1 activity in HepG2 and Huh7 cells. (A) The ACC1 activity in HepG2 cell was detected by ELISA assay. (B) The ACC1 activity in Huh7 cell was measured. * p

    Techniques Used: Activity Assay, Enzyme-linked Immunosorbent Assay

    Medium- and long-chain triglyceride (MCT/LCT) propofol inhibited free fatty acid (FFA)-induced lipid accumulation in HepG2 and Huh7 cells. (A) After FFA and 4 µg/ml or 8 µg/ml MCT/LCT propofol treatment, Oil Red O staining was used to detect the accumulation of hepatic lipids. Relative intracellular lipid level was determined in HepG2 and Huh7 cells (Oil Red O staining, magnification, ×400). (B) After 4 µg/ml or 8 µg/ml MCT/LCT propofol treatment, the TG content in different groups were detected. * p
    Figure Legend Snippet: Medium- and long-chain triglyceride (MCT/LCT) propofol inhibited free fatty acid (FFA)-induced lipid accumulation in HepG2 and Huh7 cells. (A) After FFA and 4 µg/ml or 8 µg/ml MCT/LCT propofol treatment, Oil Red O staining was used to detect the accumulation of hepatic lipids. Relative intracellular lipid level was determined in HepG2 and Huh7 cells (Oil Red O staining, magnification, ×400). (B) After 4 µg/ml or 8 µg/ml MCT/LCT propofol treatment, the TG content in different groups were detected. * p

    Techniques Used: Staining

    Cytotoxicity of free fatty acid (FFA) and medium- and long-chain triglyceride (MCT/LCT) propofol on hepatocytes. (A) HepG2 and Huh7 cells were treated with different concentrations of FFA, MTT was used to evaluate the cell viability. (B) HepG2 and Huh7 cells were treated with different concentrations of MCT/LCT propofol, then evaluated the cell viability. * p
    Figure Legend Snippet: Cytotoxicity of free fatty acid (FFA) and medium- and long-chain triglyceride (MCT/LCT) propofol on hepatocytes. (A) HepG2 and Huh7 cells were treated with different concentrations of FFA, MTT was used to evaluate the cell viability. (B) HepG2 and Huh7 cells were treated with different concentrations of MCT/LCT propofol, then evaluated the cell viability. * p

    Techniques Used: MTT Assay

    Medium- and long-chain triglyceride propofol promoted the phosphorylation of acetyl coenzyme A carboxylase (ACC) in HepG2 and Huh7 cells. (A) Western blot was carried out to detect the expression and phosphorylation of ACC in different time points and concentrations in HepG2 and Huh7 cells. Pro (µg/ml) indicates Propofol (µg/ml). (B) Relative quantitative data analyses of p-ACC expression in HepG2 and Huh7 cells. * p
    Figure Legend Snippet: Medium- and long-chain triglyceride propofol promoted the phosphorylation of acetyl coenzyme A carboxylase (ACC) in HepG2 and Huh7 cells. (A) Western blot was carried out to detect the expression and phosphorylation of ACC in different time points and concentrations in HepG2 and Huh7 cells. Pro (µg/ml) indicates Propofol (µg/ml). (B) Relative quantitative data analyses of p-ACC expression in HepG2 and Huh7 cells. * p

    Techniques Used: Western Blot, Expressing

    Medium- and long-chain triglyceride (MCT/LCT) propofol reversed free fatty acid (FFA)-induced phosphorylation inhibition of acetyl coenzyme A carboxylase (ACC) and AMPK in HepG2 and Huh7 cells. (A) Western blot was carried out to detect the expression and phosphorylation of ACC and AMPK after 4 µg/ml or 8 µg/ml MCT/LCT propofol treatment in HepG2 and Huh7 cells. (B) Relative quantitative data analyses of p-AMPK and p-ACC expression in HepG2 and Huh7 cells in different groups. * p
    Figure Legend Snippet: Medium- and long-chain triglyceride (MCT/LCT) propofol reversed free fatty acid (FFA)-induced phosphorylation inhibition of acetyl coenzyme A carboxylase (ACC) and AMPK in HepG2 and Huh7 cells. (A) Western blot was carried out to detect the expression and phosphorylation of ACC and AMPK after 4 µg/ml or 8 µg/ml MCT/LCT propofol treatment in HepG2 and Huh7 cells. (B) Relative quantitative data analyses of p-AMPK and p-ACC expression in HepG2 and Huh7 cells in different groups. * p

    Techniques Used: Inhibition, Western Blot, Expressing

    5) Product Images from "Silencing of functional p53 attenuates NAFLD by promoting HMGB1-related autophagy induction"

    Article Title: Silencing of functional p53 attenuates NAFLD by promoting HMGB1-related autophagy induction

    Journal: Hepatology International

    doi: 10.1007/s12072-020-10068-4

    Hepatic HMGB1 translocation induced by p53 silencing. a Immunochemical analysis of HMGB1 expression in livers from p53 +/+ and p53 −/− mice. b Western blotting determination of nuclear, cytoplasmic and total HMGB1 expression in p53 +/+ and p53 −/− mouse livers. c, d Western blotting determination of nuclear, cytoplasmic and total HMGB1 expression in p53-silenced c HepG2 and d Huh7 cells. H3, ACTB and GAPDH were chosen as internal controls for nuclear, cytoplasmic and total protein, respectively. * p
    Figure Legend Snippet: Hepatic HMGB1 translocation induced by p53 silencing. a Immunochemical analysis of HMGB1 expression in livers from p53 +/+ and p53 −/− mice. b Western blotting determination of nuclear, cytoplasmic and total HMGB1 expression in p53 +/+ and p53 −/− mouse livers. c, d Western blotting determination of nuclear, cytoplasmic and total HMGB1 expression in p53-silenced c HepG2 and d Huh7 cells. H3, ACTB and GAPDH were chosen as internal controls for nuclear, cytoplasmic and total protein, respectively. * p

    Techniques Used: Translocation Assay, Expressing, Mouse Assay, Western Blot

    Effect of p53 silencing on autophagy in vitro. a , b Confocal microscopy analysis of autophagosomes in a HepG2 and b Huh7 cells. Cells were first transfected with p53 siRNA for 24 h, followed by pEGFP-LC3 plasmid transfection for another 24 h. DAPI was chosen for nuclear staining. c, d Western blotting analysis of autophagy- and p53-related pathways in p53-silenced c HepG2 and d Huh7 cells. * p
    Figure Legend Snippet: Effect of p53 silencing on autophagy in vitro. a , b Confocal microscopy analysis of autophagosomes in a HepG2 and b Huh7 cells. Cells were first transfected with p53 siRNA for 24 h, followed by pEGFP-LC3 plasmid transfection for another 24 h. DAPI was chosen for nuclear staining. c, d Western blotting analysis of autophagy- and p53-related pathways in p53-silenced c HepG2 and d Huh7 cells. * p

    Techniques Used: In Vitro, Confocal Microscopy, Transfection, Plasmid Preparation, Staining, Western Blot

    p53 silencing ameliorates NAFLD symptoms in vitro. a Intracellular triglyceride levels and b ORO staining of PA-treated primary hepatocytes from p53 +/+ and p53 −/− mice (× 200). c Intracellular triglyceride levels and d ORO staining of p53-silenced HepG2 cells treated with PA (× 200). e Intracellular triglyceride levels and f ORO staining of p53-silenced Huh7 cells treated with different concentrations of PA for 24 h (× 100). * p
    Figure Legend Snippet: p53 silencing ameliorates NAFLD symptoms in vitro. a Intracellular triglyceride levels and b ORO staining of PA-treated primary hepatocytes from p53 +/+ and p53 −/− mice (× 200). c Intracellular triglyceride levels and d ORO staining of p53-silenced HepG2 cells treated with PA (× 200). e Intracellular triglyceride levels and f ORO staining of p53-silenced Huh7 cells treated with different concentrations of PA for 24 h (× 100). * p

    Techniques Used: In Vitro, Staining, Mouse Assay

    6) Product Images from "Anticancer effects of Poncirus fructus on hepatocellular carcinoma through regulation of apoptosis, migration, and invasion"

    Article Title: Anticancer effects of Poncirus fructus on hepatocellular carcinoma through regulation of apoptosis, migration, and invasion

    Journal: Oncology Reports

    doi: 10.3892/or.2020.7790

    PF induces HCC cell apoptosis. (A) Annexin V/propidium iodide (PI) staining of Hep3B and Huh7 cells after treatment with PF (20, 30 and 40 µM), as determined by fluorescence-activated cell sorting (FACS) analysis. (B) Quantitative assessment (%) of the apoptotic cells. The data represents the results from three independent experiment and expressed as mean ± SE. *P
    Figure Legend Snippet: PF induces HCC cell apoptosis. (A) Annexin V/propidium iodide (PI) staining of Hep3B and Huh7 cells after treatment with PF (20, 30 and 40 µM), as determined by fluorescence-activated cell sorting (FACS) analysis. (B) Quantitative assessment (%) of the apoptotic cells. The data represents the results from three independent experiment and expressed as mean ± SE. *P

    Techniques Used: Staining, Fluorescence, FACS

    PF inhibits the proliferation of Hep3B and Huh7 cells. (A) PF (10–40 µM) inhibited the proliferation of cells in HCC Hep3B and Huh7 as determined by the MTT assay. (B) PF (20–40 µM) inhibited the ability of Hep3B and Huh7 cell colony formation. (C) Quantitative analysis of the number of colonies in PF-treated cells compared with that of the control group. Surviving colonies with > 10 cells were counted. The data represents the results from three independent experiment and expressed as mean ± SE. **P
    Figure Legend Snippet: PF inhibits the proliferation of Hep3B and Huh7 cells. (A) PF (10–40 µM) inhibited the proliferation of cells in HCC Hep3B and Huh7 as determined by the MTT assay. (B) PF (20–40 µM) inhibited the ability of Hep3B and Huh7 cell colony formation. (C) Quantitative analysis of the number of colonies in PF-treated cells compared with that of the control group. Surviving colonies with > 10 cells were counted. The data represents the results from three independent experiment and expressed as mean ± SE. **P

    Techniques Used: MTT Assay

    PF inhibits HCC cell migration and invasion. (A and C) The effect of PF (20, 30 and 40 µM) on the migration and invasion abilities of Hep3B and Huh7 cells was examined by Transwell assays. (B and D) Quantification of the migration and invasion abilities of the Hep3B and Huh7 cells compared to the control. The chambers were stained with diff-quik stain kit, and the number of migrated or invaded cells were counted in five microscopic fields in each well at a magnification of ×200. **P
    Figure Legend Snippet: PF inhibits HCC cell migration and invasion. (A and C) The effect of PF (20, 30 and 40 µM) on the migration and invasion abilities of Hep3B and Huh7 cells was examined by Transwell assays. (B and D) Quantification of the migration and invasion abilities of the Hep3B and Huh7 cells compared to the control. The chambers were stained with diff-quik stain kit, and the number of migrated or invaded cells were counted in five microscopic fields in each well at a magnification of ×200. **P

    Techniques Used: Migration, Staining, Diff-Quik

    Effects of PF on the expression of Pin1, cyclin D1, β-catenin and EMT markers in HCC Hep3B and Huh7 cells. (A) Evaluation of Pin1, cyclin D1, β-catenin and EMT-associated protein expression by western blot analysis. (B) The protein levels of MMP-2 and MMP-9 in Hep3B and Huh7 cells after PF treatment was detected by western blot analysis. (C) The detection of pro-MMP-2 and pro-MMP-9 activity in supernatants was determined by gelatin zymography assay. PF, Poncirus fructus ; HCC, hepatocellular carcinoma; MMP, matrix metalloproteinase.
    Figure Legend Snippet: Effects of PF on the expression of Pin1, cyclin D1, β-catenin and EMT markers in HCC Hep3B and Huh7 cells. (A) Evaluation of Pin1, cyclin D1, β-catenin and EMT-associated protein expression by western blot analysis. (B) The protein levels of MMP-2 and MMP-9 in Hep3B and Huh7 cells after PF treatment was detected by western blot analysis. (C) The detection of pro-MMP-2 and pro-MMP-9 activity in supernatants was determined by gelatin zymography assay. PF, Poncirus fructus ; HCC, hepatocellular carcinoma; MMP, matrix metalloproteinase.

    Techniques Used: Expressing, Western Blot, Activity Assay, Zymography Assay

    PF induces HCC cell apoptosis by increasing ROS and disrupting mitochondrial membrane potential. (A) Western blot analysis was utilized to determine the levels of apoptosis-related proteins. (B) PF-induced changes in the mitochondrial membrane potential (ΔΨm) of Hep3B and Huh7 cells were determined with an ELISA reader after tetramethylrhodamine ethyl ester perchlorate (TMRE) staining. (C) The intracellular ROS levels in Hep3B and Huh7 cells after PF treatment. The cells were incubated with the ROS fluorescent probe, dihydroethidium (DHE), and evaluated using a confocal microscope. Scale bar, 30 µm. The data represents the results from three independent experiment and expressed as mean ± SE. ***P
    Figure Legend Snippet: PF induces HCC cell apoptosis by increasing ROS and disrupting mitochondrial membrane potential. (A) Western blot analysis was utilized to determine the levels of apoptosis-related proteins. (B) PF-induced changes in the mitochondrial membrane potential (ΔΨm) of Hep3B and Huh7 cells were determined with an ELISA reader after tetramethylrhodamine ethyl ester perchlorate (TMRE) staining. (C) The intracellular ROS levels in Hep3B and Huh7 cells after PF treatment. The cells were incubated with the ROS fluorescent probe, dihydroethidium (DHE), and evaluated using a confocal microscope. Scale bar, 30 µm. The data represents the results from three independent experiment and expressed as mean ± SE. ***P

    Techniques Used: Western Blot, Enzyme-linked Immunosorbent Assay, Staining, Incubation, Microscopy

    7) Product Images from "Direct Interaction of Human Serum Proteins with AAV Virions to Enhance AAV Transduction: Immediate Impact on Clinical Applications"

    Article Title: Direct Interaction of Human Serum Proteins with AAV Virions to Enhance AAV Transduction: Immediate Impact on Clinical Applications

    Journal: Gene therapy

    doi: 10.1038/gt.2016.75

    Human albumin increases AAV binding ability a. HSA increases AAV virus binding to Huh7 cells. AAV viruses were incubated with HSA for 2hr at 4°C, and then added to 1×10 6 Huh7 cells for 5 or 15 min at 4°C. After washing 5 times, total DNA was extracted for AAV genome copy number analysis by q-PCR. b . Imaging of liver transduction. 1×10 11 particles of AAV8/luc were administered into mice via retro-orbital vein. Twenty four hr later, the imaging was carried out and the quantitation of imaging was calculated ( c ). Forty-eight hr later, mice were euthanized and liver tissue was harvested; the luciferase activity in liver tissue lysate was measured ( d ) and the AAV genome copy number was analyzed ( e ). During the first 24hr, plasma from blood was collected at 15min, 2hr, and 24hr post AAV injection, and AAV genome copy number was analyzed ( f ). The data represented the average of 4 mice and standard deviations. (*) indicates statistically significant difference with p
    Figure Legend Snippet: Human albumin increases AAV binding ability a. HSA increases AAV virus binding to Huh7 cells. AAV viruses were incubated with HSA for 2hr at 4°C, and then added to 1×10 6 Huh7 cells for 5 or 15 min at 4°C. After washing 5 times, total DNA was extracted for AAV genome copy number analysis by q-PCR. b . Imaging of liver transduction. 1×10 11 particles of AAV8/luc were administered into mice via retro-orbital vein. Twenty four hr later, the imaging was carried out and the quantitation of imaging was calculated ( c ). Forty-eight hr later, mice were euthanized and liver tissue was harvested; the luciferase activity in liver tissue lysate was measured ( d ) and the AAV genome copy number was analyzed ( e ). During the first 24hr, plasma from blood was collected at 15min, 2hr, and 24hr post AAV injection, and AAV genome copy number was analyzed ( f ). The data represented the average of 4 mice and standard deviations. (*) indicates statistically significant difference with p

    Techniques Used: Binding Assay, Incubation, Polymerase Chain Reaction, Imaging, Transduction, Mouse Assay, Quantitation Assay, Luciferase, Activity Assay, Injection

    The enhanced effect of serum on AAV transduction a . Human serum enhances AAV transduction from different serotypes. 1×10 8 particles of AAV/luc vector were incubated with 1:500 diluted sera or PBS for 2hr at 4°C. The mixture of AAV vector and sera was used to transduce 1×10 5 Huh7 cells in a 48-well plate in the presence of adenovirus dl309 at MOI of 5. After 24 hr, luciferase activity from the cell lysate was analyzed. The fold increase of transgene expression from sera incubation was calculated by comparison to PBS. b . The effect of incubation time of AAV with human serum on enhanced transduction. 1×10 8 particles of AAV8/luc were incubated with 1:100 diluted human sera or PBS for different time periods at 4°C in the presence of ad dl309. 24hr later, luciferase expression was measured from the cell lysate. c . Enhanced AAV transduction after systemic administration. 1×10 10 particles of AAV8/luc were incubated with human serum at different dilutions for 2hr at 4°C. The mixture was administered into adult female C57BL mice via retro-orbital injection. The imaging was performed for 5min at day 3 after AAV injection. Upper panel: Representative live animal bioluminescent images of luciferase transgene expression profiles. Bottom panel: Quantification of luciferase transgene expression for enhanced AAV transduction from 6 mice after systemic administration. d . Enhanced AAV transduction after muscular injection. The mixture of AAV8/luc with human serum from Figure 1c was diluted to 1×10 9 particles/200ul in PBS and injected into mouse hind leg muscle. At week 2 post injection, the imaging was taken for 5min. Face up: left leg-AAV8 + human sera, right leg-AAV8 + PBS. Upper panel: Representative imaging. Bottom: Data of enhanced AAV transduction from 6 mice after muscular injection. The fold increase of transduction was calculated by transduction from HSA incubated AAV to that from the PBS treated one.
    Figure Legend Snippet: The enhanced effect of serum on AAV transduction a . Human serum enhances AAV transduction from different serotypes. 1×10 8 particles of AAV/luc vector were incubated with 1:500 diluted sera or PBS for 2hr at 4°C. The mixture of AAV vector and sera was used to transduce 1×10 5 Huh7 cells in a 48-well plate in the presence of adenovirus dl309 at MOI of 5. After 24 hr, luciferase activity from the cell lysate was analyzed. The fold increase of transgene expression from sera incubation was calculated by comparison to PBS. b . The effect of incubation time of AAV with human serum on enhanced transduction. 1×10 8 particles of AAV8/luc were incubated with 1:100 diluted human sera or PBS for different time periods at 4°C in the presence of ad dl309. 24hr later, luciferase expression was measured from the cell lysate. c . Enhanced AAV transduction after systemic administration. 1×10 10 particles of AAV8/luc were incubated with human serum at different dilutions for 2hr at 4°C. The mixture was administered into adult female C57BL mice via retro-orbital injection. The imaging was performed for 5min at day 3 after AAV injection. Upper panel: Representative live animal bioluminescent images of luciferase transgene expression profiles. Bottom panel: Quantification of luciferase transgene expression for enhanced AAV transduction from 6 mice after systemic administration. d . Enhanced AAV transduction after muscular injection. The mixture of AAV8/luc with human serum from Figure 1c was diluted to 1×10 9 particles/200ul in PBS and injected into mouse hind leg muscle. At week 2 post injection, the imaging was taken for 5min. Face up: left leg-AAV8 + human sera, right leg-AAV8 + PBS. Upper panel: Representative imaging. Bottom: Data of enhanced AAV transduction from 6 mice after muscular injection. The fold increase of transduction was calculated by transduction from HSA incubated AAV to that from the PBS treated one.

    Techniques Used: Transduction, Plasmid Preparation, Incubation, Luciferase, Activity Assay, Expressing, Mouse Assay, Injection, Imaging

    The effect of human albumin on AAV8 transduction a . Transduction enhancement is related to direct interaction of AAV with serum. AAV8/luc viruses were incubated with human serum or PBS at 1:100 dilution for 2 hr at 4°C, then the mixture was used to transduce Huh7 cells either in medium with FBS, serum free medium, or serum free medium plus human serum just before addition of AAV8 pre-incubated with PBS. 24 hr later, fold increase of transgene expression was calculated. b . AAV8 interaction with human albumin. 1×10 10 particles of AAV8/luc were incubated with human sera or PBS for 2hr at 4 °C, then the mixture of virus and human serum or PBS was applied to pre-Ig bound column. After washing, the column binding proteins were eluted for AAV8 genome copy number analysis. c . AAV8 transduction with albumin depleted serum. 1×10 8 particles of AAV8/luc were incubated with human serum or albumin depleted serum at different dilutions or PBS for 2hr at 4°C. Then the mixture was used to infect Huh7 cells in serum free medium. Two days later, luciferase was detected from the cell lysate, and the fold increase of transgene expression was calculated while compared to PBS. d . Recombinant human albumin enhances AAV8 transduction. AAV8/luc was incubated with recombinant human albumin (50mg/ml) or human serum at different dilutions or PBS. Transgene expression was detected 48hr later, and the fold increase of transgene expression was calculated when compared to PBS.
    Figure Legend Snippet: The effect of human albumin on AAV8 transduction a . Transduction enhancement is related to direct interaction of AAV with serum. AAV8/luc viruses were incubated with human serum or PBS at 1:100 dilution for 2 hr at 4°C, then the mixture was used to transduce Huh7 cells either in medium with FBS, serum free medium, or serum free medium plus human serum just before addition of AAV8 pre-incubated with PBS. 24 hr later, fold increase of transgene expression was calculated. b . AAV8 interaction with human albumin. 1×10 10 particles of AAV8/luc were incubated with human sera or PBS for 2hr at 4 °C, then the mixture of virus and human serum or PBS was applied to pre-Ig bound column. After washing, the column binding proteins were eluted for AAV8 genome copy number analysis. c . AAV8 transduction with albumin depleted serum. 1×10 8 particles of AAV8/luc were incubated with human serum or albumin depleted serum at different dilutions or PBS for 2hr at 4°C. Then the mixture was used to infect Huh7 cells in serum free medium. Two days later, luciferase was detected from the cell lysate, and the fold increase of transgene expression was calculated while compared to PBS. d . Recombinant human albumin enhances AAV8 transduction. AAV8/luc was incubated with recombinant human albumin (50mg/ml) or human serum at different dilutions or PBS. Transgene expression was detected 48hr later, and the fold increase of transgene expression was calculated when compared to PBS.

    Techniques Used: Transduction, Incubation, Expressing, Binding Assay, Luciferase, Recombinant

    The effect of clinical grade human albumin on AAV8 transduction a. Enhanced AAV8 transduction in Huh7 cells from clinical grade HSA. 1×10 8 particles of AAV8/luc were incubated with 5% HSA or human serum at different dilutions or PBS for 2hr at 4°C. Then the mixture was used to transduce Huh7 cells; 48hr later, luciferase expression was assayed. b . Enhanced AAV8 transduction from clinical grade HSA after systemic administration. 1×10 10 particles of AAV8/luc were incubated with 25% HSA at different dilutions and then injected into adult female C57BL mice via retro-orbital. Imaging was taken at day 7. Upper panel: Representative animal image. Bottom panel: Data of enhanced AAV transduction from 6 mice after systemic administration. c . Enhanced AAV transduction from clinical grade HSA after muscular injection. 1×10 9 particles of AAV8/luc were incubated with 25% HSA at different dilutions and then injected into muscles in C57BL mice. One week later, the imaging was performed. Upper panel: Representative animal image. Bottom: Data of enhanced AAV transduction from 6 or 7 mice after muscular injection.
    Figure Legend Snippet: The effect of clinical grade human albumin on AAV8 transduction a. Enhanced AAV8 transduction in Huh7 cells from clinical grade HSA. 1×10 8 particles of AAV8/luc were incubated with 5% HSA or human serum at different dilutions or PBS for 2hr at 4°C. Then the mixture was used to transduce Huh7 cells; 48hr later, luciferase expression was assayed. b . Enhanced AAV8 transduction from clinical grade HSA after systemic administration. 1×10 10 particles of AAV8/luc were incubated with 25% HSA at different dilutions and then injected into adult female C57BL mice via retro-orbital. Imaging was taken at day 7. Upper panel: Representative animal image. Bottom panel: Data of enhanced AAV transduction from 6 mice after systemic administration. c . Enhanced AAV transduction from clinical grade HSA after muscular injection. 1×10 9 particles of AAV8/luc were incubated with 25% HSA at different dilutions and then injected into muscles in C57BL mice. One week later, the imaging was performed. Upper panel: Representative animal image. Bottom: Data of enhanced AAV transduction from 6 or 7 mice after muscular injection.

    Techniques Used: Transduction, Incubation, Luciferase, Expressing, Injection, Mouse Assay, Imaging

    Incubation of AAV vector with HSA pre-freezing or post-thawing of viruses has the similar enhanced effect a . Enhanced transduction in Huh7 cells. 1×10 8 particles of AAV/luc were incubated with clinical grade HSA at different dilutions for 2hr at 4°C before virus freezing or after virus thawing, and then added to Huh7 cells. 48 hr later, luciferase activity in the cell lysate was measured. b and c . Enhanced muscle transduction. 1×10 9 particles of AAV8/luc were directly injected into muscles of mice. At day 7 post injection, the mouse imaging ( b ) was carried out (left panel) and the fold increase ( c ) of transgene expression was calculated (right panel, n=6). Face up: left leg-HSA, right leg-PBS.
    Figure Legend Snippet: Incubation of AAV vector with HSA pre-freezing or post-thawing of viruses has the similar enhanced effect a . Enhanced transduction in Huh7 cells. 1×10 8 particles of AAV/luc were incubated with clinical grade HSA at different dilutions for 2hr at 4°C before virus freezing or after virus thawing, and then added to Huh7 cells. 48 hr later, luciferase activity in the cell lysate was measured. b and c . Enhanced muscle transduction. 1×10 9 particles of AAV8/luc were directly injected into muscles of mice. At day 7 post injection, the mouse imaging ( b ) was carried out (left panel) and the fold increase ( c ) of transgene expression was calculated (right panel, n=6). Face up: left leg-HSA, right leg-PBS.

    Techniques Used: Incubation, Plasmid Preparation, Transduction, Luciferase, Activity Assay, Injection, Mouse Assay, Imaging, Expressing

    Interaction of human albumin with AAV doesn’t block Nab activity AAV8/luc vector was first incubated with human albumin for 2hr at 4°C, then human IVIG at different dilution was added for another 2hr at 4°C. The mixture was added to Huh7 cells. At 48hr, the transgene expression from cell lysate was measured and Nab titer was calculated. a. The effect of interaction of human albumin with AAV virions on Nab activity. b. The effect of IVIG on human albumin enhancement of AAV transduction.
    Figure Legend Snippet: Interaction of human albumin with AAV doesn’t block Nab activity AAV8/luc vector was first incubated with human albumin for 2hr at 4°C, then human IVIG at different dilution was added for another 2hr at 4°C. The mixture was added to Huh7 cells. At 48hr, the transgene expression from cell lysate was measured and Nab titer was calculated. a. The effect of interaction of human albumin with AAV virions on Nab activity. b. The effect of IVIG on human albumin enhancement of AAV transduction.

    Techniques Used: Blocking Assay, Activity Assay, Plasmid Preparation, Incubation, Expressing, Transduction

    8) Product Images from "Direct Interaction of Human Serum Proteins with AAV Virions to Enhance AAV Transduction: Immediate Impact on Clinical Applications"

    Article Title: Direct Interaction of Human Serum Proteins with AAV Virions to Enhance AAV Transduction: Immediate Impact on Clinical Applications

    Journal: Gene therapy

    doi: 10.1038/gt.2016.75

    Human albumin increases AAV binding ability a. HSA increases AAV virus binding to Huh7 cells. AAV viruses were incubated with HSA for 2hr at 4°C, and then added to 1×10 6 Huh7 cells for 5 or 15 min at 4°C. After washing 5 times, total DNA was extracted for AAV genome copy number analysis by q-PCR. b . Imaging of liver transduction. 1×10 11 particles of AAV8/luc were administered into mice via retro-orbital vein. Twenty four hr later, the imaging was carried out and the quantitation of imaging was calculated ( c ). Forty-eight hr later, mice were euthanized and liver tissue was harvested; the luciferase activity in liver tissue lysate was measured ( d ) and the AAV genome copy number was analyzed ( e ). During the first 24hr, plasma from blood was collected at 15min, 2hr, and 24hr post AAV injection, and AAV genome copy number was analyzed ( f ). The data represented the average of 4 mice and standard deviations. (*) indicates statistically significant difference with p
    Figure Legend Snippet: Human albumin increases AAV binding ability a. HSA increases AAV virus binding to Huh7 cells. AAV viruses were incubated with HSA for 2hr at 4°C, and then added to 1×10 6 Huh7 cells for 5 or 15 min at 4°C. After washing 5 times, total DNA was extracted for AAV genome copy number analysis by q-PCR. b . Imaging of liver transduction. 1×10 11 particles of AAV8/luc were administered into mice via retro-orbital vein. Twenty four hr later, the imaging was carried out and the quantitation of imaging was calculated ( c ). Forty-eight hr later, mice were euthanized and liver tissue was harvested; the luciferase activity in liver tissue lysate was measured ( d ) and the AAV genome copy number was analyzed ( e ). During the first 24hr, plasma from blood was collected at 15min, 2hr, and 24hr post AAV injection, and AAV genome copy number was analyzed ( f ). The data represented the average of 4 mice and standard deviations. (*) indicates statistically significant difference with p

    Techniques Used: Binding Assay, Incubation, Polymerase Chain Reaction, Imaging, Transduction, Mouse Assay, Quantitation Assay, Luciferase, Activity Assay, Injection

    The enhanced effect of serum on AAV transduction a . Human serum enhances AAV transduction from different serotypes. 1×10 8 particles of AAV/luc vector were incubated with 1:500 diluted sera or PBS for 2hr at 4°C. The mixture of AAV vector and sera was used to transduce 1×10 5 Huh7 cells in a 48-well plate in the presence of adenovirus dl309 at MOI of 5. After 24 hr, luciferase activity from the cell lysate was analyzed. The fold increase of transgene expression from sera incubation was calculated by comparison to PBS. b . The effect of incubation time of AAV with human serum on enhanced transduction. 1×10 8 particles of AAV8/luc were incubated with 1:100 diluted human sera or PBS for different time periods at 4°C in the presence of ad dl309. 24hr later, luciferase expression was measured from the cell lysate. c . Enhanced AAV transduction after systemic administration. 1×10 10 particles of AAV8/luc were incubated with human serum at different dilutions for 2hr at 4°C. The mixture was administered into adult female C57BL mice via retro-orbital injection. The imaging was performed for 5min at day 3 after AAV injection. Upper panel: Representative live animal bioluminescent images of luciferase transgene expression profiles. Bottom panel: Quantification of luciferase transgene expression for enhanced AAV transduction from 6 mice after systemic administration. d . Enhanced AAV transduction after muscular injection. The mixture of AAV8/luc with human serum from Figure 1c was diluted to 1×10 9 particles/200ul in PBS and injected into mouse hind leg muscle. At week 2 post injection, the imaging was taken for 5min. Face up: left leg-AAV8 + human sera, right leg-AAV8 + PBS. Upper panel: Representative imaging. Bottom: Data of enhanced AAV transduction from 6 mice after muscular injection. The fold increase of transduction was calculated by transduction from HSA incubated AAV to that from the PBS treated one.
    Figure Legend Snippet: The enhanced effect of serum on AAV transduction a . Human serum enhances AAV transduction from different serotypes. 1×10 8 particles of AAV/luc vector were incubated with 1:500 diluted sera or PBS for 2hr at 4°C. The mixture of AAV vector and sera was used to transduce 1×10 5 Huh7 cells in a 48-well plate in the presence of adenovirus dl309 at MOI of 5. After 24 hr, luciferase activity from the cell lysate was analyzed. The fold increase of transgene expression from sera incubation was calculated by comparison to PBS. b . The effect of incubation time of AAV with human serum on enhanced transduction. 1×10 8 particles of AAV8/luc were incubated with 1:100 diluted human sera or PBS for different time periods at 4°C in the presence of ad dl309. 24hr later, luciferase expression was measured from the cell lysate. c . Enhanced AAV transduction after systemic administration. 1×10 10 particles of AAV8/luc were incubated with human serum at different dilutions for 2hr at 4°C. The mixture was administered into adult female C57BL mice via retro-orbital injection. The imaging was performed for 5min at day 3 after AAV injection. Upper panel: Representative live animal bioluminescent images of luciferase transgene expression profiles. Bottom panel: Quantification of luciferase transgene expression for enhanced AAV transduction from 6 mice after systemic administration. d . Enhanced AAV transduction after muscular injection. The mixture of AAV8/luc with human serum from Figure 1c was diluted to 1×10 9 particles/200ul in PBS and injected into mouse hind leg muscle. At week 2 post injection, the imaging was taken for 5min. Face up: left leg-AAV8 + human sera, right leg-AAV8 + PBS. Upper panel: Representative imaging. Bottom: Data of enhanced AAV transduction from 6 mice after muscular injection. The fold increase of transduction was calculated by transduction from HSA incubated AAV to that from the PBS treated one.

    Techniques Used: Transduction, Plasmid Preparation, Incubation, Luciferase, Activity Assay, Expressing, Mouse Assay, Injection, Imaging

    The effect of human albumin on AAV8 transduction a . Transduction enhancement is related to direct interaction of AAV with serum. AAV8/luc viruses were incubated with human serum or PBS at 1:100 dilution for 2 hr at 4°C, then the mixture was used to transduce Huh7 cells either in medium with FBS, serum free medium, or serum free medium plus human serum just before addition of AAV8 pre-incubated with PBS. 24 hr later, fold increase of transgene expression was calculated. b . AAV8 interaction with human albumin. 1×10 10 particles of AAV8/luc were incubated with human sera or PBS for 2hr at 4 °C, then the mixture of virus and human serum or PBS was applied to pre-Ig bound column. After washing, the column binding proteins were eluted for AAV8 genome copy number analysis. c . AAV8 transduction with albumin depleted serum. 1×10 8 particles of AAV8/luc were incubated with human serum or albumin depleted serum at different dilutions or PBS for 2hr at 4°C. Then the mixture was used to infect Huh7 cells in serum free medium. Two days later, luciferase was detected from the cell lysate, and the fold increase of transgene expression was calculated while compared to PBS. d . Recombinant human albumin enhances AAV8 transduction. AAV8/luc was incubated with recombinant human albumin (50mg/ml) or human serum at different dilutions or PBS. Transgene expression was detected 48hr later, and the fold increase of transgene expression was calculated when compared to PBS.
    Figure Legend Snippet: The effect of human albumin on AAV8 transduction a . Transduction enhancement is related to direct interaction of AAV with serum. AAV8/luc viruses were incubated with human serum or PBS at 1:100 dilution for 2 hr at 4°C, then the mixture was used to transduce Huh7 cells either in medium with FBS, serum free medium, or serum free medium plus human serum just before addition of AAV8 pre-incubated with PBS. 24 hr later, fold increase of transgene expression was calculated. b . AAV8 interaction with human albumin. 1×10 10 particles of AAV8/luc were incubated with human sera or PBS for 2hr at 4 °C, then the mixture of virus and human serum or PBS was applied to pre-Ig bound column. After washing, the column binding proteins were eluted for AAV8 genome copy number analysis. c . AAV8 transduction with albumin depleted serum. 1×10 8 particles of AAV8/luc were incubated with human serum or albumin depleted serum at different dilutions or PBS for 2hr at 4°C. Then the mixture was used to infect Huh7 cells in serum free medium. Two days later, luciferase was detected from the cell lysate, and the fold increase of transgene expression was calculated while compared to PBS. d . Recombinant human albumin enhances AAV8 transduction. AAV8/luc was incubated with recombinant human albumin (50mg/ml) or human serum at different dilutions or PBS. Transgene expression was detected 48hr later, and the fold increase of transgene expression was calculated when compared to PBS.

    Techniques Used: Transduction, Incubation, Expressing, Binding Assay, Luciferase, Recombinant

    The effect of clinical grade human albumin on AAV8 transduction a. Enhanced AAV8 transduction in Huh7 cells from clinical grade HSA. 1×10 8 particles of AAV8/luc were incubated with 5% HSA or human serum at different dilutions or PBS for 2hr at 4°C. Then the mixture was used to transduce Huh7 cells; 48hr later, luciferase expression was assayed. b . Enhanced AAV8 transduction from clinical grade HSA after systemic administration. 1×10 10 particles of AAV8/luc were incubated with 25% HSA at different dilutions and then injected into adult female C57BL mice via retro-orbital. Imaging was taken at day 7. Upper panel: Representative animal image. Bottom panel: Data of enhanced AAV transduction from 6 mice after systemic administration. c . Enhanced AAV transduction from clinical grade HSA after muscular injection. 1×10 9 particles of AAV8/luc were incubated with 25% HSA at different dilutions and then injected into muscles in C57BL mice. One week later, the imaging was performed. Upper panel: Representative animal image. Bottom: Data of enhanced AAV transduction from 6 or 7 mice after muscular injection.
    Figure Legend Snippet: The effect of clinical grade human albumin on AAV8 transduction a. Enhanced AAV8 transduction in Huh7 cells from clinical grade HSA. 1×10 8 particles of AAV8/luc were incubated with 5% HSA or human serum at different dilutions or PBS for 2hr at 4°C. Then the mixture was used to transduce Huh7 cells; 48hr later, luciferase expression was assayed. b . Enhanced AAV8 transduction from clinical grade HSA after systemic administration. 1×10 10 particles of AAV8/luc were incubated with 25% HSA at different dilutions and then injected into adult female C57BL mice via retro-orbital. Imaging was taken at day 7. Upper panel: Representative animal image. Bottom panel: Data of enhanced AAV transduction from 6 mice after systemic administration. c . Enhanced AAV transduction from clinical grade HSA after muscular injection. 1×10 9 particles of AAV8/luc were incubated with 25% HSA at different dilutions and then injected into muscles in C57BL mice. One week later, the imaging was performed. Upper panel: Representative animal image. Bottom: Data of enhanced AAV transduction from 6 or 7 mice after muscular injection.

    Techniques Used: Transduction, Incubation, Luciferase, Expressing, Injection, Mouse Assay, Imaging

    Incubation of AAV vector with HSA pre-freezing or post-thawing of viruses has the similar enhanced effect a . Enhanced transduction in Huh7 cells. 1×10 8 particles of AAV/luc were incubated with clinical grade HSA at different dilutions for 2hr at 4°C before virus freezing or after virus thawing, and then added to Huh7 cells. 48 hr later, luciferase activity in the cell lysate was measured. b and c . Enhanced muscle transduction. 1×10 9 particles of AAV8/luc were directly injected into muscles of mice. At day 7 post injection, the mouse imaging ( b ) was carried out (left panel) and the fold increase ( c ) of transgene expression was calculated (right panel, n=6). Face up: left leg-HSA, right leg-PBS.
    Figure Legend Snippet: Incubation of AAV vector with HSA pre-freezing or post-thawing of viruses has the similar enhanced effect a . Enhanced transduction in Huh7 cells. 1×10 8 particles of AAV/luc were incubated with clinical grade HSA at different dilutions for 2hr at 4°C before virus freezing or after virus thawing, and then added to Huh7 cells. 48 hr later, luciferase activity in the cell lysate was measured. b and c . Enhanced muscle transduction. 1×10 9 particles of AAV8/luc were directly injected into muscles of mice. At day 7 post injection, the mouse imaging ( b ) was carried out (left panel) and the fold increase ( c ) of transgene expression was calculated (right panel, n=6). Face up: left leg-HSA, right leg-PBS.

    Techniques Used: Incubation, Plasmid Preparation, Transduction, Luciferase, Activity Assay, Injection, Mouse Assay, Imaging, Expressing

    Interaction of human albumin with AAV doesn’t block Nab activity AAV8/luc vector was first incubated with human albumin for 2hr at 4°C, then human IVIG at different dilution was added for another 2hr at 4°C. The mixture was added to Huh7 cells. At 48hr, the transgene expression from cell lysate was measured and Nab titer was calculated. a. The effect of interaction of human albumin with AAV virions on Nab activity. b. The effect of IVIG on human albumin enhancement of AAV transduction.
    Figure Legend Snippet: Interaction of human albumin with AAV doesn’t block Nab activity AAV8/luc vector was first incubated with human albumin for 2hr at 4°C, then human IVIG at different dilution was added for another 2hr at 4°C. The mixture was added to Huh7 cells. At 48hr, the transgene expression from cell lysate was measured and Nab titer was calculated. a. The effect of interaction of human albumin with AAV virions on Nab activity. b. The effect of IVIG on human albumin enhancement of AAV transduction.

    Techniques Used: Blocking Assay, Activity Assay, Plasmid Preparation, Incubation, Expressing, Transduction

    9) Product Images from "A Spiroligomer ?-Helix Mimic That Binds HDM2, Penetrates Human Cells and Stabilizes HDM2 in Cell Culture"

    Article Title: A Spiroligomer ?-Helix Mimic That Binds HDM2, Penetrates Human Cells and Stabilizes HDM2 in Cell Culture

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0045948

    Western blot analysis of HDM2, p53 and p21 in Huh7 cells as a function of concentration of spiroligomer 1 (left) or Nutlin-3 (right).
    Figure Legend Snippet: Western blot analysis of HDM2, p53 and p21 in Huh7 cells as a function of concentration of spiroligomer 1 (left) or Nutlin-3 (right).

    Techniques Used: Western Blot, Concentration Assay

    Time dependent Western analysis of spiroligomer 1 (15 µM) and Nutlin-3 (15 µM) added to Huh7 and HepG2 cells. (A) Relative HDM2 band intensity obtained when compound 1 was incubated with Huh7 cells. This plot is the average of two gels and shows a greater than 30-fold increase in the levels of HDM2. (B) Representative western gels showing the effect on HDM2 and wt-p53 in HepG2 cells in the presence of compound 1 and Nutlin-3. (C) Plotted average HDM2 band intensity relative to control (intensity of 1) as a function of time of HepG2 cells incubated with compound 1 at 15 µM. (D) Plotted average of p53 band intensity relative to control (intensity of 1) as a function of time of HepG2 cells incubated with compound 1 at 15 µM. In (C) and (D) the results of six gels (see supporting information) are averaged and standard deviations are plotted on the data. Compound 1 leads to a transient increase in HDM2 levels at 4 hours followed by a decrease. The level of p53 also increases transiently and then decreases again and the p53 changes trail the HDM2 response.
    Figure Legend Snippet: Time dependent Western analysis of spiroligomer 1 (15 µM) and Nutlin-3 (15 µM) added to Huh7 and HepG2 cells. (A) Relative HDM2 band intensity obtained when compound 1 was incubated with Huh7 cells. This plot is the average of two gels and shows a greater than 30-fold increase in the levels of HDM2. (B) Representative western gels showing the effect on HDM2 and wt-p53 in HepG2 cells in the presence of compound 1 and Nutlin-3. (C) Plotted average HDM2 band intensity relative to control (intensity of 1) as a function of time of HepG2 cells incubated with compound 1 at 15 µM. (D) Plotted average of p53 band intensity relative to control (intensity of 1) as a function of time of HepG2 cells incubated with compound 1 at 15 µM. In (C) and (D) the results of six gels (see supporting information) are averaged and standard deviations are plotted on the data. Compound 1 leads to a transient increase in HDM2 levels at 4 hours followed by a decrease. The level of p53 also increases transiently and then decreases again and the p53 changes trail the HDM2 response.

    Techniques Used: Western Blot, Incubation

    Time dependent Western analysis of the effect of compound 1 (15 µM) and nutlin-3 (15 µM) with Huh7 cells. Compound 1 causes a large increase in HDM2 levels with no effect on p53 Y220C levels. Nutlin-3 has very little effect on HDM2 and no observable effect on p53 Y220C levels.
    Figure Legend Snippet: Time dependent Western analysis of the effect of compound 1 (15 µM) and nutlin-3 (15 µM) with Huh7 cells. Compound 1 causes a large increase in HDM2 levels with no effect on p53 Y220C levels. Nutlin-3 has very little effect on HDM2 and no observable effect on p53 Y220C levels.

    Techniques Used: Western Blot

    (A) Fluorescent imaging of spiroligomer 3 in Huh7 cells (spiroligomer 3 concentration 2 µM, incubated for 24 hours, washed with phosphate-buffered saline (PBS), followed by fixation of the cells. (B) HepG2 cells that were incubated with spiroligomer 1 (0.5 mM) for 15 hours, washed with PBS and then imaged with fluorescent microscopy (concentration 0.5 µM), incubated for 15 hours and then imaged.
    Figure Legend Snippet: (A) Fluorescent imaging of spiroligomer 3 in Huh7 cells (spiroligomer 3 concentration 2 µM, incubated for 24 hours, washed with phosphate-buffered saline (PBS), followed by fixation of the cells. (B) HepG2 cells that were incubated with spiroligomer 1 (0.5 mM) for 15 hours, washed with PBS and then imaged with fluorescent microscopy (concentration 0.5 µM), incubated for 15 hours and then imaged.

    Techniques Used: Imaging, Concentration Assay, Incubation, Microscopy

    (A) Confocal imaging of spiroligomer 3 (2 µM 3, incubated for 24 hours and then washed with PBS) in live Huh7 cells. Yellow lines represent two orthogonal sections of a z-series showing the distribution of green fluorescence in the cells. (B) Optically sectioned HepG2 cells exposed to spiroligomer 1 (0.5 µM 1 , incubated for 15 hours and then washed with PBS) by confocal microscopy. Spiroligomer 1 is localized in both cytoplasmic and nuclear compartments.
    Figure Legend Snippet: (A) Confocal imaging of spiroligomer 3 (2 µM 3, incubated for 24 hours and then washed with PBS) in live Huh7 cells. Yellow lines represent two orthogonal sections of a z-series showing the distribution of green fluorescence in the cells. (B) Optically sectioned HepG2 cells exposed to spiroligomer 1 (0.5 µM 1 , incubated for 15 hours and then washed with PBS) by confocal microscopy. Spiroligomer 1 is localized in both cytoplasmic and nuclear compartments.

    Techniques Used: Imaging, Incubation, Fluorescence, Confocal Microscopy

    10) Product Images from "A novel tumor doubling time-related immune gene signature for prognosis prediction in hepatocellular carcinoma"

    Article Title: A novel tumor doubling time-related immune gene signature for prognosis prediction in hepatocellular carcinoma

    Journal: Cancer Cell International

    doi: 10.1186/s12935-021-02227-w

    Overexpressed CLEC1B inhibited cell proliferation and migration in Huh7 cells. A Lentivirus vector encoding the full-length human CLEC1B DNA sequence was used to manipulate CLEC1B expression. B CCK-8 assay, C Wound-healing assay, and D transwell assay were used to detect the effect of overexpressed CLEC1B on cell proliferation and migration ability. *p
    Figure Legend Snippet: Overexpressed CLEC1B inhibited cell proliferation and migration in Huh7 cells. A Lentivirus vector encoding the full-length human CLEC1B DNA sequence was used to manipulate CLEC1B expression. B CCK-8 assay, C Wound-healing assay, and D transwell assay were used to detect the effect of overexpressed CLEC1B on cell proliferation and migration ability. *p

    Techniques Used: Migration, Plasmid Preparation, Sequencing, Expressing, CCK-8 Assay, Wound Healing Assay, Transwell Assay

    11) Product Images from "Development of a reverse genetics system for Sosuga virus allows rapid screening of antiviral compounds"

    Article Title: Development of a reverse genetics system for Sosuga virus allows rapid screening of antiviral compounds

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0006326

    Optimization of an antiviral screening assay using a recombinant SOSV expressing ZsG. a. Schematic representation of the wild-type recombinant SOSV (rSOSV), and the reporter SOSV expressing ZsG (rSOSV/ZsG). The ZsG coding sequence was inserted immediately 5′ of the M coding sequence (antigenomic sense), separated from the viral protein by the P2A sequence from porcine teschovirus 1. This self-cleaving amino acid motif allows expression of both ZsG and M from a single mRNA generated by the parental SOSV M promoter and terminator sequences. b. Vero-E6 cells infected with either rSOSV or rSOSV/ZsG at 72 hpi showing formation of syncytia. In rSOSV/ZsG-infected cell monolayers, syncytia were associated with extensive ZsG expression (images taken at 4 × magnification). c. Growth curves for wild-type SOSV, rSOSV, and rSOSV/ZsG were performed in Vero-E6 cells infected at MOI 0.1. Titers (TCID 50 ) were determined at 24, 48, 72, 96, and 120 hpi. d. The optimized rSOSV/ZsG antiviral screening assay was validated using ribavirin. Huh7 cells were treated with a serial 2-fold dilution of ribavirin 1 h prior to infection at MOI 0.2. ZsG fluorescence (green) was measured at 72 hpi and normalized to levels in mock-treated cells (DMSO only). Cell viability (blue) was determined concurrently by measuring ATP content, with values normalized to mock-infected cells. Each point represents the mean of quadruplicate wells, with error bars showing standard deviation; graph representative of 3 independent experiments.
    Figure Legend Snippet: Optimization of an antiviral screening assay using a recombinant SOSV expressing ZsG. a. Schematic representation of the wild-type recombinant SOSV (rSOSV), and the reporter SOSV expressing ZsG (rSOSV/ZsG). The ZsG coding sequence was inserted immediately 5′ of the M coding sequence (antigenomic sense), separated from the viral protein by the P2A sequence from porcine teschovirus 1. This self-cleaving amino acid motif allows expression of both ZsG and M from a single mRNA generated by the parental SOSV M promoter and terminator sequences. b. Vero-E6 cells infected with either rSOSV or rSOSV/ZsG at 72 hpi showing formation of syncytia. In rSOSV/ZsG-infected cell monolayers, syncytia were associated with extensive ZsG expression (images taken at 4 × magnification). c. Growth curves for wild-type SOSV, rSOSV, and rSOSV/ZsG were performed in Vero-E6 cells infected at MOI 0.1. Titers (TCID 50 ) were determined at 24, 48, 72, 96, and 120 hpi. d. The optimized rSOSV/ZsG antiviral screening assay was validated using ribavirin. Huh7 cells were treated with a serial 2-fold dilution of ribavirin 1 h prior to infection at MOI 0.2. ZsG fluorescence (green) was measured at 72 hpi and normalized to levels in mock-treated cells (DMSO only). Cell viability (blue) was determined concurrently by measuring ATP content, with values normalized to mock-infected cells. Each point represents the mean of quadruplicate wells, with error bars showing standard deviation; graph representative of 3 independent experiments.

    Techniques Used: Screening Assay, Recombinant, Expressing, Sequencing, Generated, Infection, Fluorescence, Standard Deviation

    Design and optimization of the Sosuga virus minigenome screening assay. a. Genome schematic representing the design of the Sosuga virus (SOSV) minigenome segment. The minigenome contains the full-length SOSV 3′ and 5′ leader and trailer sequences, along with the gene start sequence for nucleoprotein (NP) and the gene end sequence for the viral polymerase (L), with the parental coding and intergenic regions replaced by the coding sequence of ZsGreen1 (ZsG). Transfected into cells in conjunction with plasmids expressing SOSV L, NP, and phosphoprotein (P), this minigenome allows for expression of quantifiable ZsG. b. The minigenome assay was optimized for Huh7 cells in a 96-well plate format using different ratios of the plasmids expressing L, NP, and P with a constant amount of minigenome plasmid. Cells were transfected with 75 ng minigenome plasmid and 75 ng total of plasmids expressing L, NP, and P, with ratios of each stated underneath the bars. Relative ZsG fluorescence over control reactions with no L was calculated at 48 and 72 h post transfection (hpt). c. Dose-response curve for the optimized SOSV minigenome assay against ribavirin. Cells were treated with a serial 2-fold dilution of ribavirin 1 hpt with the minigenome plasmids, and ZsG fluorescence was measured at 72 hpt. ZsG fluorescence (green) was normalized to mock-treated cells (DMSO only). Cell viability (blue) was determined concurrently by measuring ATP content, with values normalized to mock-transfected cells. Each point represents the mean of quadruplicate wells, with error bars showing standard deviation; graph is representative of 3 independent experiments.
    Figure Legend Snippet: Design and optimization of the Sosuga virus minigenome screening assay. a. Genome schematic representing the design of the Sosuga virus (SOSV) minigenome segment. The minigenome contains the full-length SOSV 3′ and 5′ leader and trailer sequences, along with the gene start sequence for nucleoprotein (NP) and the gene end sequence for the viral polymerase (L), with the parental coding and intergenic regions replaced by the coding sequence of ZsGreen1 (ZsG). Transfected into cells in conjunction with plasmids expressing SOSV L, NP, and phosphoprotein (P), this minigenome allows for expression of quantifiable ZsG. b. The minigenome assay was optimized for Huh7 cells in a 96-well plate format using different ratios of the plasmids expressing L, NP, and P with a constant amount of minigenome plasmid. Cells were transfected with 75 ng minigenome plasmid and 75 ng total of plasmids expressing L, NP, and P, with ratios of each stated underneath the bars. Relative ZsG fluorescence over control reactions with no L was calculated at 48 and 72 h post transfection (hpt). c. Dose-response curve for the optimized SOSV minigenome assay against ribavirin. Cells were treated with a serial 2-fold dilution of ribavirin 1 hpt with the minigenome plasmids, and ZsG fluorescence was measured at 72 hpt. ZsG fluorescence (green) was normalized to mock-treated cells (DMSO only). Cell viability (blue) was determined concurrently by measuring ATP content, with values normalized to mock-transfected cells. Each point represents the mean of quadruplicate wells, with error bars showing standard deviation; graph is representative of 3 independent experiments.

    Techniques Used: Screening Assay, Sequencing, Transfection, Expressing, Plasmid Preparation, Fluorescence, Standard Deviation

    Immunofluorescent imaging of rSOSV infected Huh7 cells treated with selected antiviral compounds. Huh7 cells were treated with serial dilutions of either 2′-deoxy-2′-fluorocytidine (2′-dFC), 6-azauridine, mycophenolic acid (MPA), or 09167 1 h prior to infection with rSOSV at MOI 0.2. At 48 hpi, cells were fixed and SOSV proteins (green) and cell nuclei (blue) visualized by immunofluorescence. Concentration of compound is shown in white text on each representative image (mM); control cells were treated with DMSO only. Images taken using a 20 × objective. White bar represents 100 μm.
    Figure Legend Snippet: Immunofluorescent imaging of rSOSV infected Huh7 cells treated with selected antiviral compounds. Huh7 cells were treated with serial dilutions of either 2′-deoxy-2′-fluorocytidine (2′-dFC), 6-azauridine, mycophenolic acid (MPA), or 09167 1 h prior to infection with rSOSV at MOI 0.2. At 48 hpi, cells were fixed and SOSV proteins (green) and cell nuclei (blue) visualized by immunofluorescence. Concentration of compound is shown in white text on each representative image (mM); control cells were treated with DMSO only. Images taken using a 20 × objective. White bar represents 100 μm.

    Techniques Used: Imaging, Infection, Immunofluorescence, Concentration Assay

    12) Product Images from "Host Translation Shutoff Mediated by Non-structural Protein 2 is a Critical Factor in the Antiviral State Resistance of Venezuelan Equine Encephalitis Virus"

    Article Title: Host Translation Shutoff Mediated by Non-structural Protein 2 is a Critical Factor in the Antiviral State Resistance of Venezuelan Equine Encephalitis Virus

    Journal: Virology

    doi: 10.1016/j.virol.2016.06.005

    Individually expressed viral proteins block transcription and translation (A) Huh7 cells were transfected with indicated plasmids and lysates were collected at 18h post transfection. Western blots for HA-tag were performed as described in Materials and Methods. (B and C) Huh7 cells were transfected with plasmids coding for indicated viral proteins and labeled with 100μCi/ml of [ 35 S] Cys/Met for 2h at 8-24h post transfection. Lysates were collected and resolved on SDS-PAGE gels and visualized as described in Materials and Methods. (B) Representative image of nsP and capsid induced shutoff compared to GFP control. (C) Densitometry was performed to quantify the extent of shutoff following transfection of indicated plasmids. ****, P
    Figure Legend Snippet: Individually expressed viral proteins block transcription and translation (A) Huh7 cells were transfected with indicated plasmids and lysates were collected at 18h post transfection. Western blots for HA-tag were performed as described in Materials and Methods. (B and C) Huh7 cells were transfected with plasmids coding for indicated viral proteins and labeled with 100μCi/ml of [ 35 S] Cys/Met for 2h at 8-24h post transfection. Lysates were collected and resolved on SDS-PAGE gels and visualized as described in Materials and Methods. (B) Representative image of nsP and capsid induced shutoff compared to GFP control. (C) Densitometry was performed to quantify the extent of shutoff following transfection of indicated plasmids. ****, P

    Techniques Used: Blocking Assay, Transfection, Western Blot, Labeling, SDS Page

    13) Product Images from "Coronavirus RNA synthesis takes place within membrane-bound sites"

    Article Title: Coronavirus RNA synthesis takes place within membrane-bound sites

    Journal: bioRxiv

    doi: 10.1101/2021.11.04.467246

    Viral RNA synthesis of diverse CoVs takes place within a membrane-bound compartment. (a) Huh7 cells were infected with HCoV 229E. At 7.5 hpi cells were treated with BrU and ActD for 30 min before fixation at 8 hpi. Cells were permeabilized with Triton X-100 (left) or digitonin (right) then labeled for BrU (green) and N (red), DAPI labeling the nuclei (blue). (b) VeroE6 cells were infected with SARS-CoV-2. At 5.5 hpi cells were treated with BrU and ActD for 30 min before fixation at 6 hpi. Cells were permeabilized with Triton X-100 (left) or digitonin (right) then labeled for BrU (green) and N (red), DAPI labeling the nuclei (blue). (c) LLC-PK1 cells were infected with PDCoV. At 5.5 hpi cells were treated with BrU and ActD for 30 min before fixation at 6 hpi. Cells were permeabilized with Triton X-100 (left) or digitonin (right) then labeled for BrU (green) and N (red), DAPI labeling the nuclei (blue). Scale bars represent 5 μm.
    Figure Legend Snippet: Viral RNA synthesis of diverse CoVs takes place within a membrane-bound compartment. (a) Huh7 cells were infected with HCoV 229E. At 7.5 hpi cells were treated with BrU and ActD for 30 min before fixation at 8 hpi. Cells were permeabilized with Triton X-100 (left) or digitonin (right) then labeled for BrU (green) and N (red), DAPI labeling the nuclei (blue). (b) VeroE6 cells were infected with SARS-CoV-2. At 5.5 hpi cells were treated with BrU and ActD for 30 min before fixation at 6 hpi. Cells were permeabilized with Triton X-100 (left) or digitonin (right) then labeled for BrU (green) and N (red), DAPI labeling the nuclei (blue). (c) LLC-PK1 cells were infected with PDCoV. At 5.5 hpi cells were treated with BrU and ActD for 30 min before fixation at 6 hpi. Cells were permeabilized with Triton X-100 (left) or digitonin (right) then labeled for BrU (green) and N (red), DAPI labeling the nuclei (blue). Scale bars represent 5 μm.

    Techniques Used: Infection, Labeling

    14) Product Images from "Interactomic analysis reveals a new homeostatic role for the HIV restriction factor TRIM5α in mitophagy"

    Article Title: Interactomic analysis reveals a new homeostatic role for the HIV restriction factor TRIM5α in mitophagy

    Journal: bioRxiv

    doi: 10.1101/2021.08.20.457143

    TRIM5-dependent mitophagy is independent of TRIM5 actions in HIV restriction. (A) AlamarBlue-based assessment of cell viability in WT and TRIM5 knockout Huh7 cells following 24 h treatment with the indicated concentrations of IVM. (B) The impact of infection with TRIM5-sensitive (HIV P90A) or TRIM5-resistant (HIV WT) pseudoviruses on mitochondrial protein abundance and on LC3B conversion in HeLa cells expressing YFP-Parkin. Cells were subjected to synchronous infection with VSV-G pseudotyped HIV for 24 hours prior to lysis and immunoblotting with the indicated antibodies. CCCP treatment (24 hours) was used as a positive control for mitophagy induction. (C) The impact of mitophagy-inducing compounds on the ability of RhTRIM5 to restrict HIV-1. HeLa cells stably expressing HuTRIM5 or RhTRIM5 were infected with VSV-G pseudotyped HIV-1 encoding a GFP reporter after 1.5 hours pre-treatment with CCCP, IVM, or DMSO control. Virus was then allowed to enter cells for 4 h in the presence of the compounds, after which the media was replaced. 48 h later, the percent of cells showing GFP positivity was determined by high content imaging. Data, mean ± SEM; ***, P
    Figure Legend Snippet: TRIM5-dependent mitophagy is independent of TRIM5 actions in HIV restriction. (A) AlamarBlue-based assessment of cell viability in WT and TRIM5 knockout Huh7 cells following 24 h treatment with the indicated concentrations of IVM. (B) The impact of infection with TRIM5-sensitive (HIV P90A) or TRIM5-resistant (HIV WT) pseudoviruses on mitochondrial protein abundance and on LC3B conversion in HeLa cells expressing YFP-Parkin. Cells were subjected to synchronous infection with VSV-G pseudotyped HIV for 24 hours prior to lysis and immunoblotting with the indicated antibodies. CCCP treatment (24 hours) was used as a positive control for mitophagy induction. (C) The impact of mitophagy-inducing compounds on the ability of RhTRIM5 to restrict HIV-1. HeLa cells stably expressing HuTRIM5 or RhTRIM5 were infected with VSV-G pseudotyped HIV-1 encoding a GFP reporter after 1.5 hours pre-treatment with CCCP, IVM, or DMSO control. Virus was then allowed to enter cells for 4 h in the presence of the compounds, after which the media was replaced. 48 h later, the percent of cells showing GFP positivity was determined by high content imaging. Data, mean ± SEM; ***, P

    Techniques Used: Knock-Out, Infection, Expressing, Lysis, Positive Control, Stable Transfection, Imaging

    Role of TRIM5 in Parkin-independent mitophagy. (A) Immunoblot analysis of the impact of DFP on the abundance of the indicated mitochondrial proteins in whole cell lysates from WT and TRIM5 knockout HeLa cells. Actin is used as a loading control. (B-E) Immunoblot analysis of the effect of ivermectin (IVM) treatment on the abundance of mitochondrial proteins in WT and TRIM5 knockout Huh7 cells. Cells were treated with IVM for 24 hours prior to lysis and immunoblotting with the indicated antibodies. Plots, the abundance of mitochondrial proteins COXII, VDAC1, and TOM20 was determined relative to actin. Data: *, P
    Figure Legend Snippet: Role of TRIM5 in Parkin-independent mitophagy. (A) Immunoblot analysis of the impact of DFP on the abundance of the indicated mitochondrial proteins in whole cell lysates from WT and TRIM5 knockout HeLa cells. Actin is used as a loading control. (B-E) Immunoblot analysis of the effect of ivermectin (IVM) treatment on the abundance of mitochondrial proteins in WT and TRIM5 knockout Huh7 cells. Cells were treated with IVM for 24 hours prior to lysis and immunoblotting with the indicated antibodies. Plots, the abundance of mitochondrial proteins COXII, VDAC1, and TOM20 was determined relative to actin. Data: *, P

    Techniques Used: Knock-Out, Lysis

    TRIM5 protects cells from excessive inflammation and cell death in response to mitochondrial damage. (A-D) Seahorse analysis of oxygen consumption rate (OCR) in WT and TRIM5 knockout Huh7 cells in response to ivermectin (IVM; 1.5 µM) or DMSO alone. (E) Immunoblot analysis of immune- and inflammation-related proteins in WT or TRIM5 knockout Huh7 cells following a 24 hour treatment with 15 µM IVM or DMSO control. Plots show the abundance of the indicated protein relative to a loading control (actin). Each data point represents an independent experiment. *, P
    Figure Legend Snippet: TRIM5 protects cells from excessive inflammation and cell death in response to mitochondrial damage. (A-D) Seahorse analysis of oxygen consumption rate (OCR) in WT and TRIM5 knockout Huh7 cells in response to ivermectin (IVM; 1.5 µM) or DMSO alone. (E) Immunoblot analysis of immune- and inflammation-related proteins in WT or TRIM5 knockout Huh7 cells following a 24 hour treatment with 15 µM IVM or DMSO control. Plots show the abundance of the indicated protein relative to a loading control (actin). Each data point represents an independent experiment. *, P

    Techniques Used: Knock-Out

    TRIM5 is required for mitophagy in response to mitochondrial depolarization. (A) HeLa cells stably expressing TRIM5-HA (blue) were transiently transfected with both mCherry-Parkin (red) and eGFP-DFCP1 (green) prior to 2 h CCCP treatment and confocal microscopy. Arrows indicate regions of TRIM5/DFCP1 colocalization that are associated with Parkin-labeled mitochondria. (B, C) Representative confocal images of cells treated as above and stained to detect TRIM5-HA (green), mCherry-Parkin (red) and the autophagy factors FIP200 (B) or ATG13 (C) in blue. Arrows indicate foci of colocalized green and blue signal on the outside of Parkin-decorated mitochondria. (D) Immunoblot-based analysis of mitochondrial protein abundance in lysates from WT and TRIM5 knockout Huh7 cells under basal conditions and following 24 h treatment with CCCP. The abundance of these proteins from three independent experiments are plotted relative to actin. Data: *, P
    Figure Legend Snippet: TRIM5 is required for mitophagy in response to mitochondrial depolarization. (A) HeLa cells stably expressing TRIM5-HA (blue) were transiently transfected with both mCherry-Parkin (red) and eGFP-DFCP1 (green) prior to 2 h CCCP treatment and confocal microscopy. Arrows indicate regions of TRIM5/DFCP1 colocalization that are associated with Parkin-labeled mitochondria. (B, C) Representative confocal images of cells treated as above and stained to detect TRIM5-HA (green), mCherry-Parkin (red) and the autophagy factors FIP200 (B) or ATG13 (C) in blue. Arrows indicate foci of colocalized green and blue signal on the outside of Parkin-decorated mitochondria. (D) Immunoblot-based analysis of mitochondrial protein abundance in lysates from WT and TRIM5 knockout Huh7 cells under basal conditions and following 24 h treatment with CCCP. The abundance of these proteins from three independent experiments are plotted relative to actin. Data: *, P

    Techniques Used: Stable Transfection, Expressing, Transfection, Confocal Microscopy, Labeling, Staining, Knock-Out

    TRIM5 recruits upstream autophagy regulatory machinery to damaged mitochondria. (A) Confocal microscopic analysis of LC3B and TOM20 association with WT and TRIM5 KO Huh7 cells. Arrows indicate TOM20-positive structures (mitochondria) that are enveloped within LC3B-positive signaling. Zoomed-in images of the boxed regions are shown on the right. LC3B-surrounded mitochondria could not be identified in images from TRIM5 KO Huh7 cells. (B) Recruitment of autophagy machinery to mitochondria following CCCP treatment. Lysates from mitochondria purified from WT or TRIM5 Huh7 cells treated with CCCP (20 µM) or vehicle control (DMSO) for 6 hours were subjected to immunoblotting with the indicated antibodies. Plots show the abundance of the indicated proteins in the mitochondrial fractions relative to TOM20. Data: mean + SEM; *, P
    Figure Legend Snippet: TRIM5 recruits upstream autophagy regulatory machinery to damaged mitochondria. (A) Confocal microscopic analysis of LC3B and TOM20 association with WT and TRIM5 KO Huh7 cells. Arrows indicate TOM20-positive structures (mitochondria) that are enveloped within LC3B-positive signaling. Zoomed-in images of the boxed regions are shown on the right. LC3B-surrounded mitochondria could not be identified in images from TRIM5 KO Huh7 cells. (B) Recruitment of autophagy machinery to mitochondria following CCCP treatment. Lysates from mitochondria purified from WT or TRIM5 Huh7 cells treated with CCCP (20 µM) or vehicle control (DMSO) for 6 hours were subjected to immunoblotting with the indicated antibodies. Plots show the abundance of the indicated proteins in the mitochondrial fractions relative to TOM20. Data: mean + SEM; *, P

    Techniques Used: Purification

    15) Product Images from "Paradoxical effects of cellular senescence-inhibited gene involved in hepatocellular carcinoma migration and proliferation by ERK pathway and mesenchymal-like markers"

    Article Title: Paradoxical effects of cellular senescence-inhibited gene involved in hepatocellular carcinoma migration and proliferation by ERK pathway and mesenchymal-like markers

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S188449

    The localization and expression of cellular senescence-inhibited gene (CSIG) in hepatocellular carcinoma (HCC) cells. Notes: ( A – C ) Subcellular localization of CSIG in HCC cells was examined by immunofluorescence analysis. CSIG protein was immune-labeled with anti-CSIG (Sigma-Aldrich Co.) and nucleus was counterstained with DAPI. ( A ) SMMC7721, ( B ) MHCC97H, ( C ) Huh7. ( D ) CSIG protein levels in immortalized human hepatic cell line L02, and six HCC cell lines (SMMC7721, Huh7, MHCC97L, MHCC97H, HepG2, and Bel7402) were detected by Western blotting.
    Figure Legend Snippet: The localization and expression of cellular senescence-inhibited gene (CSIG) in hepatocellular carcinoma (HCC) cells. Notes: ( A – C ) Subcellular localization of CSIG in HCC cells was examined by immunofluorescence analysis. CSIG protein was immune-labeled with anti-CSIG (Sigma-Aldrich Co.) and nucleus was counterstained with DAPI. ( A ) SMMC7721, ( B ) MHCC97H, ( C ) Huh7. ( D ) CSIG protein levels in immortalized human hepatic cell line L02, and six HCC cell lines (SMMC7721, Huh7, MHCC97L, MHCC97H, HepG2, and Bel7402) were detected by Western blotting.

    Techniques Used: Expressing, Immunofluorescence, Labeling, Western Blot

    Schematic diagram demonstrating the molecular pathway in two cellular senescence-inhibited gene (CSIG) overexpressing cell lines (SMMC7721-CSIG and Huh7-CSIG). Notes: CSIG obviously activated P-ERK cascade and mesenchymal cell markers, thereby increasing proliferation and migration capacities of SMMC7721 cells (left panel). CSIG obviously inhibited P-ERK cascade as well as protein levels of mesenchymal cell markers, thereby suppressing proliferation and migration capacities of Huh7 cells (right panel).
    Figure Legend Snippet: Schematic diagram demonstrating the molecular pathway in two cellular senescence-inhibited gene (CSIG) overexpressing cell lines (SMMC7721-CSIG and Huh7-CSIG). Notes: CSIG obviously activated P-ERK cascade and mesenchymal cell markers, thereby increasing proliferation and migration capacities of SMMC7721 cells (left panel). CSIG obviously inhibited P-ERK cascade as well as protein levels of mesenchymal cell markers, thereby suppressing proliferation and migration capacities of Huh7 cells (right panel).

    Techniques Used: Migration

    Cellular senescence-inhibited gene (CSIG) suppressed proliferative and migratory capabilities of Huh7 cells. Notes: ( A ) RT-PCR and Western blotting assays showed that CSIG mRNA and protein levels were overexpressed in Huh7 cells transfected with PIRES-flag-plasmids, respectively. ( B ) Growth properties of Huh7-control and Huh7-CSIG were analyzed by viability assays. ( C , D ) The migratory capability of Huh7 cells was markedly decreased with PIRES-flag-CSIG plasmids transfection. *** P
    Figure Legend Snippet: Cellular senescence-inhibited gene (CSIG) suppressed proliferative and migratory capabilities of Huh7 cells. Notes: ( A ) RT-PCR and Western blotting assays showed that CSIG mRNA and protein levels were overexpressed in Huh7 cells transfected with PIRES-flag-plasmids, respectively. ( B ) Growth properties of Huh7-control and Huh7-CSIG were analyzed by viability assays. ( C , D ) The migratory capability of Huh7 cells was markedly decreased with PIRES-flag-CSIG plasmids transfection. *** P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection

    Cellular senescence-inhibited gene (CSIG) could inhibit P-ERK activation and levels of mesenchymal-like markers in Huh7 cells. Notes: ( A ) Western blot showed the protein levels of P-ERK in Huh7 cells transfected with control vector (PIRES-flag) and CSIG overexpressed plasmid (PIRES-flag-CSIG). ( B ) The mRNA levels of CSIG, E-cadherin, Vimentin, N-cadherin, and MMP9 in Huh7 cells transfected with CSIG overexpressed plasmid and control vector. ( C , D ) The protein levels of E-cadherin, Vimentin, N-cadherin, and MMP9 in Huh7 cells transfected with CSIG overexpressed plasmid and control vector. ** P
    Figure Legend Snippet: Cellular senescence-inhibited gene (CSIG) could inhibit P-ERK activation and levels of mesenchymal-like markers in Huh7 cells. Notes: ( A ) Western blot showed the protein levels of P-ERK in Huh7 cells transfected with control vector (PIRES-flag) and CSIG overexpressed plasmid (PIRES-flag-CSIG). ( B ) The mRNA levels of CSIG, E-cadherin, Vimentin, N-cadherin, and MMP9 in Huh7 cells transfected with CSIG overexpressed plasmid and control vector. ( C , D ) The protein levels of E-cadherin, Vimentin, N-cadherin, and MMP9 in Huh7 cells transfected with CSIG overexpressed plasmid and control vector. ** P

    Techniques Used: Activation Assay, Western Blot, Transfection, Plasmid Preparation

    16) Product Images from "Selection of a Clinical Lead TCR Targeting Alpha-Fetoprotein-Positive Liver Cancer Based on a Balance of Risk and Benefit"

    Article Title: Selection of a Clinical Lead TCR Targeting Alpha-Fetoprotein-Positive Liver Cancer Based on a Balance of Risk and Benefit

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.00623

    AFP TCR 2 shows lower than background activity toward primary neurons and no alloreactivity compared to TCR 1 and 3. (A) IFN-γ concentration in the supernatant of overnight, 1:1 ratio co-culture of the indicated AFP TCR T cells with iCell derived neurons measured by ELISA. Data represents the mean + s.d. of the triplicate co-cultures and is representative of T cells prepared from 2 healthy donors. (B) IFN-γ concentration in the supernatant of overnight, 1:1 ratio co-culture of the indicated AFP TCR T cells with a panel of Epstein-Barr virus transformed B cell lines expressing various HLA alleles measured by ELISA. Data represents the mean + s.d. of quadruplicate co-cultures and is representative of T cells prepared from 2 healthy donors. HepG2 and Huh7 cells were included as positive and negative controls, respectively, in both (A,B) . * P
    Figure Legend Snippet: AFP TCR 2 shows lower than background activity toward primary neurons and no alloreactivity compared to TCR 1 and 3. (A) IFN-γ concentration in the supernatant of overnight, 1:1 ratio co-culture of the indicated AFP TCR T cells with iCell derived neurons measured by ELISA. Data represents the mean + s.d. of the triplicate co-cultures and is representative of T cells prepared from 2 healthy donors. (B) IFN-γ concentration in the supernatant of overnight, 1:1 ratio co-culture of the indicated AFP TCR T cells with a panel of Epstein-Barr virus transformed B cell lines expressing various HLA alleles measured by ELISA. Data represents the mean + s.d. of quadruplicate co-cultures and is representative of T cells prepared from 2 healthy donors. HepG2 and Huh7 cells were included as positive and negative controls, respectively, in both (A,B) . * P

    Techniques Used: Activity Assay, Concentration Assay, Co-Culture Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay, Transformation Assay, Expressing

    AFP TCR1, 2, and 3 demonstrate comparable potent reactivity toward the target cells in vitro . (A) Alignment of amino acid sequence of CDR3 regions of the α (left) and β (right) chains of the 7 AFP TCRs. (B) FACS analysis of the surface expression of TCR β chain and AFP tetramer staining of T cells 8 days after transducing of indicated AFP TCRs. The percentage of TCR β (top) or AFP tetramer (bottom) positive cells as indicated. (C) Cytokine production and degranulation of AFP TCR T cells upon encountering HepG2 target cells as revealed by intracellular staining of IL-2, IFN-γ (top), TNF-α and CD107a (bottom). (D) IFN-γ concentration in the supernatant of overnight, 1:1 ratio co-culture of AFP TCR T cells with HepG2 target cells or control Huh7 cells measured by ELISA. Data represents the mean + s.d. of quadruplicate co-culture samples. (E) Cytolytic capacity of AFP TCR T cells toward HepG2 target cells over a 4-day co-culture. Data represents the mean of triplicate samples derived from RTCA instrument, as compared to target only wells. (E:T): effect to target ratio. Colors are represented as in (D) . (F) Time to eradicate 50% of target cells (KT50) of different AFP TCR T cells at the indicated effect to target (E:T) ratio. Colors are represented as in (D) . T cells transduced with TCR 8 and TCR 11 did not eradicate 50% of the target by the end of the analysis at E:T = 0.25:1, thus were not plotted. Data shown in b-f is representative of at least 3 independent experiments on T cells isolated from 3 healthy donors. UT, untransduced T cells.
    Figure Legend Snippet: AFP TCR1, 2, and 3 demonstrate comparable potent reactivity toward the target cells in vitro . (A) Alignment of amino acid sequence of CDR3 regions of the α (left) and β (right) chains of the 7 AFP TCRs. (B) FACS analysis of the surface expression of TCR β chain and AFP tetramer staining of T cells 8 days after transducing of indicated AFP TCRs. The percentage of TCR β (top) or AFP tetramer (bottom) positive cells as indicated. (C) Cytokine production and degranulation of AFP TCR T cells upon encountering HepG2 target cells as revealed by intracellular staining of IL-2, IFN-γ (top), TNF-α and CD107a (bottom). (D) IFN-γ concentration in the supernatant of overnight, 1:1 ratio co-culture of AFP TCR T cells with HepG2 target cells or control Huh7 cells measured by ELISA. Data represents the mean + s.d. of quadruplicate co-culture samples. (E) Cytolytic capacity of AFP TCR T cells toward HepG2 target cells over a 4-day co-culture. Data represents the mean of triplicate samples derived from RTCA instrument, as compared to target only wells. (E:T): effect to target ratio. Colors are represented as in (D) . (F) Time to eradicate 50% of target cells (KT50) of different AFP TCR T cells at the indicated effect to target (E:T) ratio. Colors are represented as in (D) . T cells transduced with TCR 8 and TCR 11 did not eradicate 50% of the target by the end of the analysis at E:T = 0.25:1, thus were not plotted. Data shown in b-f is representative of at least 3 independent experiments on T cells isolated from 3 healthy donors. UT, untransduced T cells.

    Techniques Used: In Vitro, Sequencing, FACS, Expressing, Staining, Concentration Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Transduction, Isolation

    AFP TCR 2 displays no reactivity toward a variety of HLA-A*02:01 + primary cells. (A) IFN-γ concentration in the supernatant of overnight, 1:1 ratio co-culture of UT or T cells expressing AFP TCR 2 with primary hepatocytes measured by ELISA. T cells were prepared from 2 healthy donors and human primary hepatocytes were obtained from 2 adult HLA-A*02:01 + donors. Data represents the mean + s.d. of quadruplicate co-cultures. (B) 4-1BB upregulation by T cells after co-culture as described in a. Cells were further analyzed by FACS for surface expression of 4-1BB on total CD3 + untransduced T cells or AFP TCR 2 transduced T cells. HepG2 and Huh7 cells were included as positive and negative controls, respectively, in both (A,B) . (C) Summary of reactivity from co-culture experiment of T cells expressing AFP TCR 2 with primary hepatocytes, primary lung and kidney epithelial cells, and iCell derived primary cells including neurons, astrocytes, cardiomyocyte, and endothelial cells.
    Figure Legend Snippet: AFP TCR 2 displays no reactivity toward a variety of HLA-A*02:01 + primary cells. (A) IFN-γ concentration in the supernatant of overnight, 1:1 ratio co-culture of UT or T cells expressing AFP TCR 2 with primary hepatocytes measured by ELISA. T cells were prepared from 2 healthy donors and human primary hepatocytes were obtained from 2 adult HLA-A*02:01 + donors. Data represents the mean + s.d. of quadruplicate co-cultures. (B) 4-1BB upregulation by T cells after co-culture as described in a. Cells were further analyzed by FACS for surface expression of 4-1BB on total CD3 + untransduced T cells or AFP TCR 2 transduced T cells. HepG2 and Huh7 cells were included as positive and negative controls, respectively, in both (A,B) . (C) Summary of reactivity from co-culture experiment of T cells expressing AFP TCR 2 with primary hepatocytes, primary lung and kidney epithelial cells, and iCell derived primary cells including neurons, astrocytes, cardiomyocyte, and endothelial cells.

    Techniques Used: Concentration Assay, Co-Culture Assay, Expressing, Enzyme-linked Immunosorbent Assay, FACS, Derivative Assay

    17) Product Images from "Glucagon-like Peptide-1 Receptor (GLP-1R) is present on human hepatocytes and has a direct role in decreasing hepatic steatosis in vitro by modulating elements of the insulin signaling pathway"

    Article Title: Glucagon-like Peptide-1 Receptor (GLP-1R) is present on human hepatocytes and has a direct role in decreasing hepatic steatosis in vitro by modulating elements of the insulin signaling pathway

    Journal: Hepatology (Baltimore, Md.)

    doi: 10.1002/hep.23569

    Knockdown of GLP-1R by siRNA HuH7 cells were transfected with siRNA (at 30nM) against GlP-1R, and Western blot analysis with β-actin serving as loading control was performed. A ) Knockdown was achieved as compared to control with 30nM siGLP-1R. B) Transfected HuH7 cells were treated with Exendin-4 (10nM) for 60 min. siRNA GLP-1R abolished the Exendin-4 mediated-effects on PDK-1 and PKC-ζ. ( B and C, respectively ). These studies represent multiple independent experiments. (* p
    Figure Legend Snippet: Knockdown of GLP-1R by siRNA HuH7 cells were transfected with siRNA (at 30nM) against GlP-1R, and Western blot analysis with β-actin serving as loading control was performed. A ) Knockdown was achieved as compared to control with 30nM siGLP-1R. B) Transfected HuH7 cells were treated with Exendin-4 (10nM) for 60 min. siRNA GLP-1R abolished the Exendin-4 mediated-effects on PDK-1 and PKC-ζ. ( B and C, respectively ). These studies represent multiple independent experiments. (* p

    Techniques Used: Transfection, Western Blot

    Reduction of steatosis on exposure to Exendin-4 A HuH7 cells were treated with palmitic acid ( 400uM/l) and oleic acid ( 400uM/l) for 12 h under insulin-free conditions; and subsequently exposed to Exendin-4 (20nM) for 6 h. Figure 3a) shows Oil red O staining of HuH7 cells treated with FFA and Exendin-4. A marked increase in Oil red O stained droplets (red) are visible in the cells treated with FFA as compared with the non treated cells. On exposure to Exendin4 there is a significant loss of fat droplets (40X). B : Triglyceride assay was performed on HuH7 cell lysate after treatment with palmitic and oleic acid followed by exposure to Exendin-4 as described in MATERIALS AND METHODS. Bars show % increase in TG content and then % decrease on treatment with Exendin-4. (Means ± SE; * p
    Figure Legend Snippet: Reduction of steatosis on exposure to Exendin-4 A HuH7 cells were treated with palmitic acid ( 400uM/l) and oleic acid ( 400uM/l) for 12 h under insulin-free conditions; and subsequently exposed to Exendin-4 (20nM) for 6 h. Figure 3a) shows Oil red O staining of HuH7 cells treated with FFA and Exendin-4. A marked increase in Oil red O stained droplets (red) are visible in the cells treated with FFA as compared with the non treated cells. On exposure to Exendin4 there is a significant loss of fat droplets (40X). B : Triglyceride assay was performed on HuH7 cell lysate after treatment with palmitic and oleic acid followed by exposure to Exendin-4 as described in MATERIALS AND METHODS. Bars show % increase in TG content and then % decrease on treatment with Exendin-4. (Means ± SE; * p

    Techniques Used: Staining

    Identification of GLP-1R on hepatocytes A Primary human hepatocyte and HuH7 cells show the presence of GLP-1R by western blot analysis. Brain lysate was used as a positive control. B: Bioluminescence analysis for GPCRs demonstrates the presence of GLP-1R on HuH7 cells. Bars show % increase in bioluminescence in GLP-1R as compared with no- primary-antibody treatment. (Means ± SE; * p
    Figure Legend Snippet: Identification of GLP-1R on hepatocytes A Primary human hepatocyte and HuH7 cells show the presence of GLP-1R by western blot analysis. Brain lysate was used as a positive control. B: Bioluminescence analysis for GPCRs demonstrates the presence of GLP-1R on HuH7 cells. Bars show % increase in bioluminescence in GLP-1R as compared with no- primary-antibody treatment. (Means ± SE; * p

    Techniques Used: Western Blot, Positive Control

    18) Product Images from "RAB1B interacts with TRAF3 to promote antiviral innate immunity"

    Article Title: RAB1B interacts with TRAF3 to promote antiviral innate immunity

    Journal: bioRxiv

    doi: 10.1101/542050

    RAB1B is required for full induction of IFN-β in response to RIG-I pathway activation by SenV. A , RT-qPCR analysis of IFNB1 , relative to GAPDH and normalized to the Vector, Mock sample, in 293T cells treated with control (CTL) or RAB1B siRNAs for 24 h and then Mock-or SenV-infected (8 h), along with immunoblot analysis for RAB1B expression. B , RT-qPCR analysis for IFNB1 , relative to GAPDH and normalized to the WT, Mock sample, in WT or RAB1B KO 293T cells infected with SenV (20 h). For A and B , individual dots represent technical replicates with bars displaying the mean ± SD of one of three representative experiments. C, Focus forming assay of ZIKV focus-forming units (FFU) from supernatants of Huh7 cells infected with ZIKV (48 hours post infection, MOI 0.01) after transfection of CTL or RAB1B siRNAs (48 h), measured as the percentage of FFU relative to siCTL. Individual dots represent biological replicates with bars displaying the mean ± SEM (n=3). ** P≤0.01, *** P≤0.001, by an unpaired Student’s t-test comparing SenV samples ( A-B ) and or siRAB1B to siCTL ( C ).
    Figure Legend Snippet: RAB1B is required for full induction of IFN-β in response to RIG-I pathway activation by SenV. A , RT-qPCR analysis of IFNB1 , relative to GAPDH and normalized to the Vector, Mock sample, in 293T cells treated with control (CTL) or RAB1B siRNAs for 24 h and then Mock-or SenV-infected (8 h), along with immunoblot analysis for RAB1B expression. B , RT-qPCR analysis for IFNB1 , relative to GAPDH and normalized to the WT, Mock sample, in WT or RAB1B KO 293T cells infected with SenV (20 h). For A and B , individual dots represent technical replicates with bars displaying the mean ± SD of one of three representative experiments. C, Focus forming assay of ZIKV focus-forming units (FFU) from supernatants of Huh7 cells infected with ZIKV (48 hours post infection, MOI 0.01) after transfection of CTL or RAB1B siRNAs (48 h), measured as the percentage of FFU relative to siCTL. Individual dots represent biological replicates with bars displaying the mean ± SEM (n=3). ** P≤0.01, *** P≤0.001, by an unpaired Student’s t-test comparing SenV samples ( A-B ) and or siRAB1B to siCTL ( C ).

    Techniques Used: Activation Assay, Quantitative RT-PCR, Plasmid Preparation, Infection, Expressing, Focus Forming Assay, Transfection

    19) Product Images from "Novel and potent inhibitors targeting DHODH, a rate-limiting enzyme in de novo pyrimidine biosynthesis, are broad-spectrum antiviral against RNA viruses including newly emerged coronavirus SARS-CoV-2"

    Article Title: Novel and potent inhibitors targeting DHODH, a rate-limiting enzyme in de novo pyrimidine biosynthesis, are broad-spectrum antiviral against RNA viruses including newly emerged coronavirus SARS-CoV-2

    Journal: bioRxiv

    doi: 10.1101/2020.03.11.983056

    Broad-spectrum antiviral activities of DHODH inhibitors. (A) Anti-Ebola replication efficacy. BSR-T7/5 cells were transfected with the EBOV mini-genome replication system (NP, VP35, VP30, MG, and L) in the presence of increasing concentrations of Teriflunomide, Brequinar, S312 and S416 respectively. Inhibitory effects of these compounds (EC 50 ) to EBOV mini-genome replication were determined using Bright-Glo Luciferase Assay (left-hand scale, red curve). CC 50 of compounds were determined by analyzing BSR-T7/5 cell viability using CellTiterGlo Assay (right-hand scale, green curve). The results are presented as a mean of at least two replicates ± SD. (B) Anti-Zika virus efficacy. Huh7 cells were infected with Zika virus (MOI=0.05) for 4 hours and then treated with increasing concentrations of compounds Teriflunomide, Brequinar, S312 and S416 respectively. The viral yields in cell supernatants were then quantified by qRT-PCR to reflect the replication efficiency of Zika virus. (C) Anti-SARS-CoV-2 virus efficacy. Aliquots of Vero E6 cells were seeded in 96-well plates and then infected with Beta CoV/Wuhan/WIV04/2019 at MOI of 0.03. At the same time, different concentrations of the compounds were added for co-culture. Cell supernatants were harvested 48 h.p.i. and RNA was extracted and quantified by qRT-PCR to determine the numbers of viral RNA copies. (D) Immuno-fluorescence assay of SARS-CoV-2-infected cells. Vero E6 cells were infected with SARS-CoV-2 under the same procedure of C. Cells were fixed and permeabilized for staining with anti-viral NP antibody, followed by staining with Alexa 488-labeled secondary antibody. Green represents infected cells. Nuclei were stained by DAPI, and the merge of NP and nuclei were shown. Scale bar, 400uM. The results (B, C) are presented as a mean of at least three replicates ± SD. Statistical analysis, One-way ANOVA for (B). NS, p > 0.05; *, p
    Figure Legend Snippet: Broad-spectrum antiviral activities of DHODH inhibitors. (A) Anti-Ebola replication efficacy. BSR-T7/5 cells were transfected with the EBOV mini-genome replication system (NP, VP35, VP30, MG, and L) in the presence of increasing concentrations of Teriflunomide, Brequinar, S312 and S416 respectively. Inhibitory effects of these compounds (EC 50 ) to EBOV mini-genome replication were determined using Bright-Glo Luciferase Assay (left-hand scale, red curve). CC 50 of compounds were determined by analyzing BSR-T7/5 cell viability using CellTiterGlo Assay (right-hand scale, green curve). The results are presented as a mean of at least two replicates ± SD. (B) Anti-Zika virus efficacy. Huh7 cells were infected with Zika virus (MOI=0.05) for 4 hours and then treated with increasing concentrations of compounds Teriflunomide, Brequinar, S312 and S416 respectively. The viral yields in cell supernatants were then quantified by qRT-PCR to reflect the replication efficiency of Zika virus. (C) Anti-SARS-CoV-2 virus efficacy. Aliquots of Vero E6 cells were seeded in 96-well plates and then infected with Beta CoV/Wuhan/WIV04/2019 at MOI of 0.03. At the same time, different concentrations of the compounds were added for co-culture. Cell supernatants were harvested 48 h.p.i. and RNA was extracted and quantified by qRT-PCR to determine the numbers of viral RNA copies. (D) Immuno-fluorescence assay of SARS-CoV-2-infected cells. Vero E6 cells were infected with SARS-CoV-2 under the same procedure of C. Cells were fixed and permeabilized for staining with anti-viral NP antibody, followed by staining with Alexa 488-labeled secondary antibody. Green represents infected cells. Nuclei were stained by DAPI, and the merge of NP and nuclei were shown. Scale bar, 400uM. The results (B, C) are presented as a mean of at least three replicates ± SD. Statistical analysis, One-way ANOVA for (B). NS, p > 0.05; *, p

    Techniques Used: Transfection, Luciferase, Infection, Quantitative RT-PCR, Co-Culture Assay, Fluorescence, Staining, Labeling

    20) Product Images from "miR-106b promotes cancer progression in hepatitis B virus-associated hepatocellular carcinoma"

    Article Title: miR-106b promotes cancer progression in hepatitis B virus-associated hepatocellular carcinoma

    Journal: World Journal of Gastroenterology

    doi: 10.3748/wjg.v22.i22.5183

    The mRNA expression levels of miR-106b-25 cluster and MCM7 in hepatitis B virus X protein-transfected hepatocellular carcinoma cell lines ( n = 3). The hepatitis B virus X protein (HBx) protein expression plasmid was transiently transfected into Huh7 and
    Figure Legend Snippet: The mRNA expression levels of miR-106b-25 cluster and MCM7 in hepatitis B virus X protein-transfected hepatocellular carcinoma cell lines ( n = 3). The hepatitis B virus X protein (HBx) protein expression plasmid was transiently transfected into Huh7 and

    Techniques Used: Expressing, Transfection, Plasmid Preparation

    21) Product Images from "Sar1 Affects the Localization of Perilipin 2 to Lipid Droplets"

    Article Title: Sar1 Affects the Localization of Perilipin 2 to Lipid Droplets

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms23126366

    PLIN2 localization to lipid droplets attenuated in Sar1 depleted cells. HuH7 cells treated with shRNA scramble (Scr), sh SAR1B , sh SAR1A , or sh SAR1B and SAR1A , were incubated with (OA) or without (Con) 0.5 mM oleic acid for 24 h. ( A ) Total RNA was isolated from cells. RT-PCR was performed to determine the mRNA levels of the SAR1A and SAR1B . The ratio of SAR1 mRNA level relative to the GAPDH mRNA level is shown in arbitrary units. SAR1 mRNA levels in OA-treated cells were set to 1.0. The results are presented as the mean ± SD of three independent experiments. a p
    Figure Legend Snippet: PLIN2 localization to lipid droplets attenuated in Sar1 depleted cells. HuH7 cells treated with shRNA scramble (Scr), sh SAR1B , sh SAR1A , or sh SAR1B and SAR1A , were incubated with (OA) or without (Con) 0.5 mM oleic acid for 24 h. ( A ) Total RNA was isolated from cells. RT-PCR was performed to determine the mRNA levels of the SAR1A and SAR1B . The ratio of SAR1 mRNA level relative to the GAPDH mRNA level is shown in arbitrary units. SAR1 mRNA levels in OA-treated cells were set to 1.0. The results are presented as the mean ± SD of three independent experiments. a p

    Techniques Used: shRNA, Incubation, Isolation, Reverse Transcription Polymerase Chain Reaction

    Effect of Sar1a on PLIN2 localization to lipid droplets. HuH7 cells transiently expressing GFP-Sar1a WT or GFP-Sar1a H79G were treated with 0.5 mM oleic acid (OA) for 24 h. ( A ) Lysates from cells containing LDs were fractionated by sucrose density gradient centrifugation, and an aliquot (10%) of each fraction was analyzed by western blotting using the antibodies against the indicated proteins. ( B ) The ratio of PLIN2 intensity in LD fractions to sum of all fractions are shown as a scattered dot plot. *** p
    Figure Legend Snippet: Effect of Sar1a on PLIN2 localization to lipid droplets. HuH7 cells transiently expressing GFP-Sar1a WT or GFP-Sar1a H79G were treated with 0.5 mM oleic acid (OA) for 24 h. ( A ) Lysates from cells containing LDs were fractionated by sucrose density gradient centrifugation, and an aliquot (10%) of each fraction was analyzed by western blotting using the antibodies against the indicated proteins. ( B ) The ratio of PLIN2 intensity in LD fractions to sum of all fractions are shown as a scattered dot plot. *** p

    Techniques Used: Expressing, Gradient Centrifugation, Western Blot

    Partial colocalization between PLIN2 and GFP-Sar1b WT in lipid droplets. HuH7 cells transiently expressing GFP ( a ), GFP-Sar1b WT ( b ), GFP-Sar1b H79G ( c ) were treated with ( B : OA) or without ( A : Con) 0.5 mM oleic acid for 24 h. The cells were stained with the antibody against PLIN2 and DAPI. Boxed areas are shown in higher magnification in the insets. Bar, 10 μm. Data are representative of experiments repeated more than three times.
    Figure Legend Snippet: Partial colocalization between PLIN2 and GFP-Sar1b WT in lipid droplets. HuH7 cells transiently expressing GFP ( a ), GFP-Sar1b WT ( b ), GFP-Sar1b H79G ( c ) were treated with ( B : OA) or without ( A : Con) 0.5 mM oleic acid for 24 h. The cells were stained with the antibody against PLIN2 and DAPI. Boxed areas are shown in higher magnification in the insets. Bar, 10 μm. Data are representative of experiments repeated more than three times.

    Techniques Used: Expressing, Staining

    Peripheral localization of GFP-Sar1b WT on lipid droplet in oleic acid-treated cells. ( A ) HuH7 cells transiently expressing GFP ( a ) GFP-Sar1b WT ( b ) GFP-Sar1b H79G ( c ) were treated with (OA) or without (Con) 0.5 mM oleic acid for 24 h. The cells were stained with LipidTox and DAPI. Boxed areas are shown in higher magnification in the insets. Bar, 10 μm. Data are representative of experiments repeated at least three times. ( B ) The number of LDs per area in ( A ) are shown. Results are shown as a scattered dot plot. p -Values for GFP Con vs. GFP-Sar1b WT Con; and GFP OA vs. GFP-Sar1b WT OA were 0.350 and 0.417, respectively. ( C ) The ratio of LD area to total cell area in ( A ) are shown.
    Figure Legend Snippet: Peripheral localization of GFP-Sar1b WT on lipid droplet in oleic acid-treated cells. ( A ) HuH7 cells transiently expressing GFP ( a ) GFP-Sar1b WT ( b ) GFP-Sar1b H79G ( c ) were treated with (OA) or without (Con) 0.5 mM oleic acid for 24 h. The cells were stained with LipidTox and DAPI. Boxed areas are shown in higher magnification in the insets. Bar, 10 μm. Data are representative of experiments repeated at least three times. ( B ) The number of LDs per area in ( A ) are shown. Results are shown as a scattered dot plot. p -Values for GFP Con vs. GFP-Sar1b WT Con; and GFP OA vs. GFP-Sar1b WT OA were 0.350 and 0.417, respectively. ( C ) The ratio of LD area to total cell area in ( A ) are shown.

    Techniques Used: Expressing, Staining

    The intracellular PLIN2 protein levels decrease in cells expressing GFP-Sar1b H79G. HuH7 cells transiently expressing GFP, GFP-Sar1b WT, or GFP-Sar1b H79G were treated with (OA) or without (Con) 0.5 mM oleic acid for 24 h. ( A ) Intracellular triacylglycerol (TG) levels were quantified. Results are presented as a scattered dot plot. ( B , C ) Cell lysates were analyzed by western blotting using antibodies against the indicated proteins. Data are representative of experiments repeated at least three times. ( D ) The ratio of PLIN2 protein levels relative to GAPDH is expressed in arbitrary units. The PLIN2 levels in OA-treated cells expressing GFP were set to 1.0. The results are presented as a scattered dot plot. ** p
    Figure Legend Snippet: The intracellular PLIN2 protein levels decrease in cells expressing GFP-Sar1b H79G. HuH7 cells transiently expressing GFP, GFP-Sar1b WT, or GFP-Sar1b H79G were treated with (OA) or without (Con) 0.5 mM oleic acid for 24 h. ( A ) Intracellular triacylglycerol (TG) levels were quantified. Results are presented as a scattered dot plot. ( B , C ) Cell lysates were analyzed by western blotting using antibodies against the indicated proteins. Data are representative of experiments repeated at least three times. ( D ) The ratio of PLIN2 protein levels relative to GAPDH is expressed in arbitrary units. The PLIN2 levels in OA-treated cells expressing GFP were set to 1.0. The results are presented as a scattered dot plot. ** p

    Techniques Used: Expressing, Western Blot

    22) Product Images from "Host Translation Shutoff Mediated by Non-structural Protein 2 is a Critical Factor in the Antiviral State Resistance of Venezuelan Equine Encephalitis Virus"

    Article Title: Host Translation Shutoff Mediated by Non-structural Protein 2 is a Critical Factor in the Antiviral State Resistance of Venezuelan Equine Encephalitis Virus

    Journal: Virology

    doi: 10.1016/j.virol.2016.06.005

    Individually expressed viral proteins block transcription and translation (A) Huh7 cells were transfected with indicated plasmids and lysates were collected at 18h post transfection. Western blots for HA-tag were performed as described in Materials and Methods. (B and C) Huh7 cells were transfected with plasmids coding for indicated viral proteins and labeled with 100μCi/ml of [ 35 S] Cys/Met for 2h at 8-24h post transfection. Lysates were collected and resolved on SDS-PAGE gels and visualized as described in Materials and Methods. (B) Representative image of nsP and capsid induced shutoff compared to GFP control. (C) Densitometry was performed to quantify the extent of shutoff following transfection of indicated plasmids. ****, P
    Figure Legend Snippet: Individually expressed viral proteins block transcription and translation (A) Huh7 cells were transfected with indicated plasmids and lysates were collected at 18h post transfection. Western blots for HA-tag were performed as described in Materials and Methods. (B and C) Huh7 cells were transfected with plasmids coding for indicated viral proteins and labeled with 100μCi/ml of [ 35 S] Cys/Met for 2h at 8-24h post transfection. Lysates were collected and resolved on SDS-PAGE gels and visualized as described in Materials and Methods. (B) Representative image of nsP and capsid induced shutoff compared to GFP control. (C) Densitometry was performed to quantify the extent of shutoff following transfection of indicated plasmids. ****, P

    Techniques Used: Blocking Assay, Transfection, Western Blot, Labeling, SDS Page

    23) Product Images from "Cell-Type-Dependent Regulation of mTORC1 by REDD1 and the Tumor Suppressors TSC1/TSC2 and LKB1 in Response to Hypoxia ▿"

    Article Title: Cell-Type-Dependent Regulation of mTORC1 by REDD1 and the Tumor Suppressors TSC1/TSC2 and LKB1 in Response to Hypoxia ▿

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01393-10

    AMPK and raptor phosphorylation are essential for mTORC1 inhibition by hypoxia in liver cells. (A) Western blot of Huh7 cells transfected with the indicated siRNAs (or a scrambled control [Sc]) and exposed to hypoxia for the indicated number of hours. (B) Western blot of Huh7 cells pretreated (+) with compound C (Comp C) and exposed to hypoxia for the indicated number of hours. (C) Western blot of Huh7 cells stably transduced with AMPK dominant-negative (DN) mutants (T172A [TA] or K45R [KR] mutant) or an empty vector (EV) control and exposed to hypoxia (1% O 2 ) (H) or 2-deoxyglucose (2DG) (normoxia [N]). (D) Western blots of Huh7 cells in which endogenous raptor has been replaced by a phosphorylation-deficient raptor S722A/S792A (AA) or by a wild-type raptor control (WT).
    Figure Legend Snippet: AMPK and raptor phosphorylation are essential for mTORC1 inhibition by hypoxia in liver cells. (A) Western blot of Huh7 cells transfected with the indicated siRNAs (or a scrambled control [Sc]) and exposed to hypoxia for the indicated number of hours. (B) Western blot of Huh7 cells pretreated (+) with compound C (Comp C) and exposed to hypoxia for the indicated number of hours. (C) Western blot of Huh7 cells stably transduced with AMPK dominant-negative (DN) mutants (T172A [TA] or K45R [KR] mutant) or an empty vector (EV) control and exposed to hypoxia (1% O 2 ) (H) or 2-deoxyglucose (2DG) (normoxia [N]). (D) Western blots of Huh7 cells in which endogenous raptor has been replaced by a phosphorylation-deficient raptor S722A/S792A (AA) or by a wild-type raptor control (WT).

    Techniques Used: Inhibition, Western Blot, Transfection, Stable Transfection, Transduction, Dominant Negative Mutation, Mutagenesis, Plasmid Preparation

    24) Product Images from "Development of a reverse genetics system for Sosuga virus allows rapid screening of antiviral compounds"

    Article Title: Development of a reverse genetics system for Sosuga virus allows rapid screening of antiviral compounds

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0006326

    Optimization of an antiviral screening assay using a recombinant SOSV expressing ZsG. a. Schematic representation of the wild-type recombinant SOSV (rSOSV), and the reporter SOSV expressing ZsG (rSOSV/ZsG). The ZsG coding sequence was inserted immediately 5′ of the M coding sequence (antigenomic sense), separated from the viral protein by the P2A sequence from porcine teschovirus 1. This self-cleaving amino acid motif allows expression of both ZsG and M from a single mRNA generated by the parental SOSV M promoter and terminator sequences. b. Vero-E6 cells infected with either rSOSV or rSOSV/ZsG at 72 hpi showing formation of syncytia. In rSOSV/ZsG-infected cell monolayers, syncytia were associated with extensive ZsG expression (images taken at 4 × magnification). c. Growth curves for wild-type SOSV, rSOSV, and rSOSV/ZsG were performed in Vero-E6 cells infected at MOI 0.1. Titers (TCID 50 ) were determined at 24, 48, 72, 96, and 120 hpi. d. The optimized rSOSV/ZsG antiviral screening assay was validated using ribavirin. Huh7 cells were treated with a serial 2-fold dilution of ribavirin 1 h prior to infection at MOI 0.2. ZsG fluorescence (green) was measured at 72 hpi and normalized to levels in mock-treated cells (DMSO only). Cell viability (blue) was determined concurrently by measuring ATP content, with values normalized to mock-infected cells. Each point represents the mean of quadruplicate wells, with error bars showing standard deviation; graph representative of 3 independent experiments.
    Figure Legend Snippet: Optimization of an antiviral screening assay using a recombinant SOSV expressing ZsG. a. Schematic representation of the wild-type recombinant SOSV (rSOSV), and the reporter SOSV expressing ZsG (rSOSV/ZsG). The ZsG coding sequence was inserted immediately 5′ of the M coding sequence (antigenomic sense), separated from the viral protein by the P2A sequence from porcine teschovirus 1. This self-cleaving amino acid motif allows expression of both ZsG and M from a single mRNA generated by the parental SOSV M promoter and terminator sequences. b. Vero-E6 cells infected with either rSOSV or rSOSV/ZsG at 72 hpi showing formation of syncytia. In rSOSV/ZsG-infected cell monolayers, syncytia were associated with extensive ZsG expression (images taken at 4 × magnification). c. Growth curves for wild-type SOSV, rSOSV, and rSOSV/ZsG were performed in Vero-E6 cells infected at MOI 0.1. Titers (TCID 50 ) were determined at 24, 48, 72, 96, and 120 hpi. d. The optimized rSOSV/ZsG antiviral screening assay was validated using ribavirin. Huh7 cells were treated with a serial 2-fold dilution of ribavirin 1 h prior to infection at MOI 0.2. ZsG fluorescence (green) was measured at 72 hpi and normalized to levels in mock-treated cells (DMSO only). Cell viability (blue) was determined concurrently by measuring ATP content, with values normalized to mock-infected cells. Each point represents the mean of quadruplicate wells, with error bars showing standard deviation; graph representative of 3 independent experiments.

    Techniques Used: Screening Assay, Recombinant, Expressing, Sequencing, Generated, Infection, Fluorescence, Standard Deviation

    Design and optimization of the Sosuga virus minigenome screening assay. a. Genome schematic representing the design of the Sosuga virus (SOSV) minigenome segment. The minigenome contains the full-length SOSV 3′ and 5′ leader and trailer sequences, along with the gene start sequence for nucleoprotein (NP) and the gene end sequence for the viral polymerase (L), with the parental coding and intergenic regions replaced by the coding sequence of ZsGreen1 (ZsG). Transfected into cells in conjunction with plasmids expressing SOSV L, NP, and phosphoprotein (P), this minigenome allows for expression of quantifiable ZsG. b. The minigenome assay was optimized for Huh7 cells in a 96-well plate format using different ratios of the plasmids expressing L, NP, and P with a constant amount of minigenome plasmid. Cells were transfected with 75 ng minigenome plasmid and 75 ng total of plasmids expressing L, NP, and P, with ratios of each stated underneath the bars. Relative ZsG fluorescence over control reactions with no L was calculated at 48 and 72 h post transfection (hpt). c. Dose-response curve for the optimized SOSV minigenome assay against ribavirin. Cells were treated with a serial 2-fold dilution of ribavirin 1 hpt with the minigenome plasmids, and ZsG fluorescence was measured at 72 hpt. ZsG fluorescence (green) was normalized to mock-treated cells (DMSO only). Cell viability (blue) was determined concurrently by measuring ATP content, with values normalized to mock-transfected cells. Each point represents the mean of quadruplicate wells, with error bars showing standard deviation; graph is representative of 3 independent experiments.
    Figure Legend Snippet: Design and optimization of the Sosuga virus minigenome screening assay. a. Genome schematic representing the design of the Sosuga virus (SOSV) minigenome segment. The minigenome contains the full-length SOSV 3′ and 5′ leader and trailer sequences, along with the gene start sequence for nucleoprotein (NP) and the gene end sequence for the viral polymerase (L), with the parental coding and intergenic regions replaced by the coding sequence of ZsGreen1 (ZsG). Transfected into cells in conjunction with plasmids expressing SOSV L, NP, and phosphoprotein (P), this minigenome allows for expression of quantifiable ZsG. b. The minigenome assay was optimized for Huh7 cells in a 96-well plate format using different ratios of the plasmids expressing L, NP, and P with a constant amount of minigenome plasmid. Cells were transfected with 75 ng minigenome plasmid and 75 ng total of plasmids expressing L, NP, and P, with ratios of each stated underneath the bars. Relative ZsG fluorescence over control reactions with no L was calculated at 48 and 72 h post transfection (hpt). c. Dose-response curve for the optimized SOSV minigenome assay against ribavirin. Cells were treated with a serial 2-fold dilution of ribavirin 1 hpt with the minigenome plasmids, and ZsG fluorescence was measured at 72 hpt. ZsG fluorescence (green) was normalized to mock-treated cells (DMSO only). Cell viability (blue) was determined concurrently by measuring ATP content, with values normalized to mock-transfected cells. Each point represents the mean of quadruplicate wells, with error bars showing standard deviation; graph is representative of 3 independent experiments.

    Techniques Used: Screening Assay, Sequencing, Transfection, Expressing, Plasmid Preparation, Fluorescence, Standard Deviation

    Immunofluorescent imaging of rSOSV infected Huh7 cells treated with selected antiviral compounds. Huh7 cells were treated with serial dilutions of either 2′-deoxy-2′-fluorocytidine (2′-dFC), 6-azauridine, mycophenolic acid (MPA), or 09167 1 h prior to infection with rSOSV at MOI 0.2. At 48 hpi, cells were fixed and SOSV proteins (green) and cell nuclei (blue) visualized by immunofluorescence. Concentration of compound is shown in white text on each representative image (mM); control cells were treated with DMSO only. Images taken using a 20 × objective. White bar represents 100 μm.
    Figure Legend Snippet: Immunofluorescent imaging of rSOSV infected Huh7 cells treated with selected antiviral compounds. Huh7 cells were treated with serial dilutions of either 2′-deoxy-2′-fluorocytidine (2′-dFC), 6-azauridine, mycophenolic acid (MPA), or 09167 1 h prior to infection with rSOSV at MOI 0.2. At 48 hpi, cells were fixed and SOSV proteins (green) and cell nuclei (blue) visualized by immunofluorescence. Concentration of compound is shown in white text on each representative image (mM); control cells were treated with DMSO only. Images taken using a 20 × objective. White bar represents 100 μm.

    Techniques Used: Imaging, Infection, Immunofluorescence, Concentration Assay

    25) Product Images from "Statins Suppress Ebola Virus Infectivity by Interfering with Glycoprotein Processing"

    Article Title: Statins Suppress Ebola Virus Infectivity by Interfering with Glycoprotein Processing

    Journal: mBio

    doi: 10.1128/mBio.00660-18

    Statin inhibits specific infectivity of EBOV but does not affect entry. (A) Huh7 cells pretreated with the indicated concentrations of statin or DMSO for 48 h were infected with EBOV (MOI = 3.0). For a positive control, cells were pretreated for 1 h with various concentrations of U18666A before infection with EBOV. After 1 h, infected cells were washed with serum-free medium; fresh medium with or without statin or U18666A was then added back to the cells. After 3 h of incubation at 37°C, NP gene RNA copy numbers were determined by qRT-PCR and normalized to GAPDH mRNA. Results represent mean percent normalized NP RNA levels, with error bars indicating standard deviations calculated from 3 independent experiments. *, P
    Figure Legend Snippet: Statin inhibits specific infectivity of EBOV but does not affect entry. (A) Huh7 cells pretreated with the indicated concentrations of statin or DMSO for 48 h were infected with EBOV (MOI = 3.0). For a positive control, cells were pretreated for 1 h with various concentrations of U18666A before infection with EBOV. After 1 h, infected cells were washed with serum-free medium; fresh medium with or without statin or U18666A was then added back to the cells. After 3 h of incubation at 37°C, NP gene RNA copy numbers were determined by qRT-PCR and normalized to GAPDH mRNA. Results represent mean percent normalized NP RNA levels, with error bars indicating standard deviations calculated from 3 independent experiments. *, P

    Techniques Used: Infection, Positive Control, Incubation, Quantitative RT-PCR

    Antiviral activity of other statins against EBOV. (A) Huh7 cells were infected with Ebola virus (EBOV) at an MOI of 0.05. After infection, cells were washed and then treated with various concentrations of lovastatin, fluvastatin, simvastatin, atorvastatin, rosuvastatin, and pitavastatin or DMSO (control). Culture supernatants were harvested 72 hpi, and viral titers were quantified by 50% tissue culture infective dose (TCID 50 ) determination. (B) Viability (percentage) of Huh7 cells treated with lovastatin, fluvastatin, simvastatin, atorvastatin, rosuvastatin, and pitavastatin or DMSO (control) was determined after 72 h of treatment. Values were normalized to DMSO-treated controls.
    Figure Legend Snippet: Antiviral activity of other statins against EBOV. (A) Huh7 cells were infected with Ebola virus (EBOV) at an MOI of 0.05. After infection, cells were washed and then treated with various concentrations of lovastatin, fluvastatin, simvastatin, atorvastatin, rosuvastatin, and pitavastatin or DMSO (control). Culture supernatants were harvested 72 hpi, and viral titers were quantified by 50% tissue culture infective dose (TCID 50 ) determination. (B) Viability (percentage) of Huh7 cells treated with lovastatin, fluvastatin, simvastatin, atorvastatin, rosuvastatin, and pitavastatin or DMSO (control) was determined after 72 h of treatment. Values were normalized to DMSO-treated controls.

    Techniques Used: Activity Assay, Infection

    Statin inhibits EBOV GP processing. (A) Huh7 cells were transfected with a plasmid expressing EBOV GP 1,2 and treated with statin or DMSO. GP and actin expression in cell lysates was then analyzed by Western blotting. To determine the extent of GP 1 cleavage, blot images were subjected to densitometry analysis. The percentage of GP 1 was determined by dividing the signal of GP 1 over the total amount of glycoprotein recognized by GP 1 MAb [GP 1 /(GP 1 + preGP)]. (B) Cell lysates of EBOV GP 1,2 -transfected cells either were left untreated or were digested with endo H or PNGase. GP and actin expression was then visualized by Western blotting. (C) Huh7 cells transfected with a plasmid expressing EBOV GP 1,2 were treated with 50 µM statin or DMSO in the presence of various concentrations of mevalonate. GP and actin expression in cell lysates was analyzed by Western blotting. The percentage of GP 1 was determined as in panel A. (D) Huh7 cells were transfected with a plasmid expressing either wild-type EBOV GP 1,2 or GP 1,2 in which the furin cleavage motif (furin-site mutant GP 1,2 ) had been mutated and were treated with statin (left panel), proprotein convertase inhibitor (PC inh, right panel), or DMSO control. GP and actin expression in cell lysates was then analyzed by Western blotting. To determine the extent of GP 0 cleavage, blot images were subjected to densitometry analysis. Percentage of GP 1 or GP 0 was determined by dividing the signal of GP 1 or GP 0 over the total amount of glycoprotein recognized by GP 1,0 MAb [GP 1,0 /(GP 1,0 + preGP)].
    Figure Legend Snippet: Statin inhibits EBOV GP processing. (A) Huh7 cells were transfected with a plasmid expressing EBOV GP 1,2 and treated with statin or DMSO. GP and actin expression in cell lysates was then analyzed by Western blotting. To determine the extent of GP 1 cleavage, blot images were subjected to densitometry analysis. The percentage of GP 1 was determined by dividing the signal of GP 1 over the total amount of glycoprotein recognized by GP 1 MAb [GP 1 /(GP 1 + preGP)]. (B) Cell lysates of EBOV GP 1,2 -transfected cells either were left untreated or were digested with endo H or PNGase. GP and actin expression was then visualized by Western blotting. (C) Huh7 cells transfected with a plasmid expressing EBOV GP 1,2 were treated with 50 µM statin or DMSO in the presence of various concentrations of mevalonate. GP and actin expression in cell lysates was analyzed by Western blotting. The percentage of GP 1 was determined as in panel A. (D) Huh7 cells were transfected with a plasmid expressing either wild-type EBOV GP 1,2 or GP 1,2 in which the furin cleavage motif (furin-site mutant GP 1,2 ) had been mutated and were treated with statin (left panel), proprotein convertase inhibitor (PC inh, right panel), or DMSO control. GP and actin expression in cell lysates was then analyzed by Western blotting. To determine the extent of GP 0 cleavage, blot images were subjected to densitometry analysis. Percentage of GP 1 or GP 0 was determined by dividing the signal of GP 1 or GP 0 over the total amount of glycoprotein recognized by GP 1,0 MAb [GP 1,0 /(GP 1,0 + preGP)].

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Western Blot, Mutagenesis

    Statin inhibits GP processing and incorporation of GP 1 into EBOV particles. Huh7 cells infected with EBOV at an MOI of 2.0 were treated with DMSO or statin. Supernatants and cell lysates were collected, and EBOV particles were purified from supernatants by ultracentrifugation through a sucrose cushion. Purified EBOV particles were resuspended in 2× sample lysis buffer, and levels of GP and VP40 were analyzed by Western blotting. Percentage of GP 1 was determined by dividing the signal of GP 1 over the total amount of glycoprotein recognized by GP 1 MAb [GP 1 /(GP 1 + preGP)].
    Figure Legend Snippet: Statin inhibits GP processing and incorporation of GP 1 into EBOV particles. Huh7 cells infected with EBOV at an MOI of 2.0 were treated with DMSO or statin. Supernatants and cell lysates were collected, and EBOV particles were purified from supernatants by ultracentrifugation through a sucrose cushion. Purified EBOV particles were resuspended in 2× sample lysis buffer, and levels of GP and VP40 were analyzed by Western blotting. Percentage of GP 1 was determined by dividing the signal of GP 1 over the total amount of glycoprotein recognized by GP 1 MAb [GP 1 /(GP 1 + preGP)].

    Techniques Used: Infection, Purification, Lysis, Western Blot

    Statin inhibits Ebola virus infection. (A) Huh7 cells were infected with Ebola virus (EBOV) at an MOI of 0.05. After infection, cells were washed and then treated with various concentrations of statin or with DMSO (control). At 72 hpi, the cells were fixed, permeabilized, and stained with anti-EBOV rabbit polyclonal antibody. (B) Culture supernatants of Huh7 cells infected with EBOV and treated with statin or DMSO as in panel A were harvested 72 hpi, and viral titers were quantified by 50% tissue culture infective dose (TCID 50 ) determination. (C) Viability (percent) of statin-treated Huh7 cells was determined after 72 h of treatment. Values were normalized to DMSO-treated controls. (D) Human monocyte-derived macrophages from 4 separate donors were infected with EBOV at an MOI of 0.05, and cells were washed and then treated with various concentrations of statin or DMSO. Cell supernatants were harvested 72 hpi, and viral titers were quantified by TCID 50 determination. The results shown are means ± standard deviations from triplicate wells and representative of two independent experiments. (E) Viability (percent) of statin-treated and mock-infected human monocytes/macrophages was determined after 72 h of treatment. Values were normalized to DMSO controls.
    Figure Legend Snippet: Statin inhibits Ebola virus infection. (A) Huh7 cells were infected with Ebola virus (EBOV) at an MOI of 0.05. After infection, cells were washed and then treated with various concentrations of statin or with DMSO (control). At 72 hpi, the cells were fixed, permeabilized, and stained with anti-EBOV rabbit polyclonal antibody. (B) Culture supernatants of Huh7 cells infected with EBOV and treated with statin or DMSO as in panel A were harvested 72 hpi, and viral titers were quantified by 50% tissue culture infective dose (TCID 50 ) determination. (C) Viability (percent) of statin-treated Huh7 cells was determined after 72 h of treatment. Values were normalized to DMSO-treated controls. (D) Human monocyte-derived macrophages from 4 separate donors were infected with EBOV at an MOI of 0.05, and cells were washed and then treated with various concentrations of statin or DMSO. Cell supernatants were harvested 72 hpi, and viral titers were quantified by TCID 50 determination. The results shown are means ± standard deviations from triplicate wells and representative of two independent experiments. (E) Viability (percent) of statin-treated and mock-infected human monocytes/macrophages was determined after 72 h of treatment. Values were normalized to DMSO controls.

    Techniques Used: Infection, Staining, Derivative Assay

    26) Product Images from "Inhibiting IRE1α-endonuclease activity decreases tumor burden in a mouse model for hepatocellular carcinoma"

    Article Title: Inhibiting IRE1α-endonuclease activity decreases tumor burden in a mouse model for hepatocellular carcinoma

    Journal: eLife

    doi: 10.7554/eLife.55865

    Inhibiting IRE1α alters generation of ROS. ( A ) intracellular ROS-levels in LX2, HepG2, and Huh7 cells treated with 50 μM 4μ8C, 100 μM 4μ8C or controls. ( B ) intracellular ROS-levels in LX2, HepG2 and Huh7 cells transfected with IRE1α-siRNA (si-IRE1α) or mock-transfected (Scr). p-Values were calculated via the Student's T-test from three biological replicates per group.
    Figure Legend Snippet: Inhibiting IRE1α alters generation of ROS. ( A ) intracellular ROS-levels in LX2, HepG2, and Huh7 cells treated with 50 μM 4μ8C, 100 μM 4μ8C or controls. ( B ) intracellular ROS-levels in LX2, HepG2 and Huh7 cells transfected with IRE1α-siRNA (si-IRE1α) or mock-transfected (Scr). p-Values were calculated via the Student's T-test from three biological replicates per group.

    Techniques Used: Transfection

    Protein levels of IRE1a in mock transfected and si-IRE1a transfected Huh7 cells.
    Figure Legend Snippet: Protein levels of IRE1a in mock transfected and si-IRE1a transfected Huh7 cells.

    Techniques Used: Transfection

    Inhibition of IRE1α decreases tumor cell proliferation. ( A ) PCNA mRNA-expression of HepG2 or Huh7-cells grown with LX2-cells in transwell inserts and treated with the IRE1α-inhibitor 4μ8C or control. ( B ) Relative cell number of LX2 and HepG2 or ( C ) LX2 and Huh7-cells treated with 4μ8C or control. ( D ) Representative images of tumor cells (HepG2 or Huh7) and LX2-stellate cells stained with antibodies against the HCC-marker EPCAM and the proliferation marker KI67. ( E ) Cell proliferation of HepG2 or HepG2+LX2 spheroids and ( F ) Huh7 or Huh7+LX2 spheroids treated with 4μ8C or control. p-Values were calculated via the Student's T-test from nine biological replicates per group, scale bars = 50 μm.
    Figure Legend Snippet: Inhibition of IRE1α decreases tumor cell proliferation. ( A ) PCNA mRNA-expression of HepG2 or Huh7-cells grown with LX2-cells in transwell inserts and treated with the IRE1α-inhibitor 4μ8C or control. ( B ) Relative cell number of LX2 and HepG2 or ( C ) LX2 and Huh7-cells treated with 4μ8C or control. ( D ) Representative images of tumor cells (HepG2 or Huh7) and LX2-stellate cells stained with antibodies against the HCC-marker EPCAM and the proliferation marker KI67. ( E ) Cell proliferation of HepG2 or HepG2+LX2 spheroids and ( F ) Huh7 or Huh7+LX2 spheroids treated with 4μ8C or control. p-Values were calculated via the Student's T-test from nine biological replicates per group, scale bars = 50 μm.

    Techniques Used: Inhibition, Expressing, Staining, Marker

    Secretion of TGFβ by tumor cells activates stellate cells and induces ER-stress. ( A ) Concentration of TGFβ in medium from tumor cells (HepG2 or Huh7) grown in mono-culture or co-cultured with LX2-stellate cells, treated with 4μ8C or control. ( B ) Concentration of TGFβ in medium from liver scaffolds engrafted with stellate cells ( C ) (LX2) and tumor cells (HepG2) treated with 4μ8C or control. mRNA-expression of the ER-stress markers DDIT3, ( D ) spliced XBP1, ( E ) unspliced XBP1 and ( F ) HSPA5 in hepatic stellate cells (LX2) grown as mono-culture or in co-cultures with the cancer cell lines HepG2 and Huh7 treated with the TGFβ receptor inhibitor SB-431541 or control. ( G ) mRNA-expression of stellate cell activation markers ACTA2 and ( H ) collagen in LX2-cells grown with HepG2 or Huh7-cells and treated with SB-431541 or control. p-Values were calculated via the Student's T-test from seven biological replicates per group.
    Figure Legend Snippet: Secretion of TGFβ by tumor cells activates stellate cells and induces ER-stress. ( A ) Concentration of TGFβ in medium from tumor cells (HepG2 or Huh7) grown in mono-culture or co-cultured with LX2-stellate cells, treated with 4μ8C or control. ( B ) Concentration of TGFβ in medium from liver scaffolds engrafted with stellate cells ( C ) (LX2) and tumor cells (HepG2) treated with 4μ8C or control. mRNA-expression of the ER-stress markers DDIT3, ( D ) spliced XBP1, ( E ) unspliced XBP1 and ( F ) HSPA5 in hepatic stellate cells (LX2) grown as mono-culture or in co-cultures with the cancer cell lines HepG2 and Huh7 treated with the TGFβ receptor inhibitor SB-431541 or control. ( G ) mRNA-expression of stellate cell activation markers ACTA2 and ( H ) collagen in LX2-cells grown with HepG2 or Huh7-cells and treated with SB-431541 or control. p-Values were calculated via the Student's T-test from seven biological replicates per group.

    Techniques Used: Concentration Assay, Cell Culture, Expressing, Activation Assay

    27) Product Images from "Nuclear lamina genetic variants, including a truncated LAP2, in twins and siblings with nonalcoholic fatty liver disease"

    Article Title: Nuclear lamina genetic variants, including a truncated LAP2, in twins and siblings with nonalcoholic fatty liver disease

    Journal: Hepatology (Baltimore, Md.)

    doi: 10.1002/hep.29522

    The truncated variant of LAP2 is mislocalized in transfected cells. (A) Huh7 cells were transfected with full-length (F.L.) LAP2α or truncated LAP2 (1-99) with DDK tag, or empty vector. Cells were then fixed, and LAP2 was visualized by indirect immunofluorescence using anti-FLAG antibody (which recognizes the DDK tag). Scale bar: 50 μm (lower magnification images: first, second, and fourth rows), 20 μm (higher magnification images: third and fifth rows). (B) Cells transfected as in panel A were scored according to whether LAP2 was specifically localized to the nucleus or mislocalized throughout the cell (represented as percent of transfected cells with mislocalized LAP2). Data were derived from counting 9–12 high-power fields from three independent experiments. Error bars represent the standard error of the mean. Student’s t test was used to determine statistical significance at a threshold of P
    Figure Legend Snippet: The truncated variant of LAP2 is mislocalized in transfected cells. (A) Huh7 cells were transfected with full-length (F.L.) LAP2α or truncated LAP2 (1-99) with DDK tag, or empty vector. Cells were then fixed, and LAP2 was visualized by indirect immunofluorescence using anti-FLAG antibody (which recognizes the DDK tag). Scale bar: 50 μm (lower magnification images: first, second, and fourth rows), 20 μm (higher magnification images: third and fifth rows). (B) Cells transfected as in panel A were scored according to whether LAP2 was specifically localized to the nucleus or mislocalized throughout the cell (represented as percent of transfected cells with mislocalized LAP2). Data were derived from counting 9–12 high-power fields from three independent experiments. Error bars represent the standard error of the mean. Student’s t test was used to determine statistical significance at a threshold of P

    Techniques Used: Variant Assay, Transfection, Plasmid Preparation, Immunofluorescence, Derivative Assay

    Variants of LAP2 found in NAFLD patients interfere with binding to lamin A. (A) Huh7 cells were co-transfected with myc-tagged wild-type (WT) or variant LAP2α and GFP-tagged lamin A, followed by immunoprecipitation using an antibody directed to the GFP tag. Immunoprecipitates were resolved on SDS-PAGE, and precipitated proteins were visualized after immunoblotting with antibodies to myc or the GFP tag. (B) Huh7 cells were co-transfected with GFP-tagged lamin A and WT LAP2α or its truncated variant (LAP2 1-99). Lamin A was immunoprecipitated, followed by visualization as in panel A. Ig, immunoglobulin; M.W., apparent molecular weight; kDa, kilodaltons; IP, immunoprecipitation; IB, immunoblot. Arrowhead indicates a non-specific band recognized by the myc antibody.
    Figure Legend Snippet: Variants of LAP2 found in NAFLD patients interfere with binding to lamin A. (A) Huh7 cells were co-transfected with myc-tagged wild-type (WT) or variant LAP2α and GFP-tagged lamin A, followed by immunoprecipitation using an antibody directed to the GFP tag. Immunoprecipitates were resolved on SDS-PAGE, and precipitated proteins were visualized after immunoblotting with antibodies to myc or the GFP tag. (B) Huh7 cells were co-transfected with GFP-tagged lamin A and WT LAP2α or its truncated variant (LAP2 1-99). Lamin A was immunoprecipitated, followed by visualization as in panel A. Ig, immunoglobulin; M.W., apparent molecular weight; kDa, kilodaltons; IP, immunoprecipitation; IB, immunoblot. Arrowhead indicates a non-specific band recognized by the myc antibody.

    Techniques Used: Binding Assay, Transfection, Variant Assay, Immunoprecipitation, SDS Page, Molecular Weight

    Truncated LAP2 has multiple unique cytoplasmic interaction partners, including p62/SQSTM1. (A) Huh7 cells were transfected with GFP-tagged LAP2α or truncated LAP2 (1-99), followed by immunoprecipitation using an anti-GFP antibody; empty vector (GFP only) and GFP-tagged lamin A were included as controls. The immunoprecipitates and the input cell lysates were visualized by silver staining. (B) Huh7 cells were transfected with GFP alone, GFP-tagged LAP2α, or truncated LAP2 (1-99), followed by immunoprecipitation of LAP2 using anti-GFP antibody. Co-precipitated p62/SQSTM1 was visualized by immunoblotting. (C) Immunoprecipitated GFP and GFP-tagged proteins were visualized by Coomassie staining for the samples shown in panel B. M.W., apparent molecular weight; kDa, kilodaltons; IP, immunoprecipitates. Arrowheads indicate antibody heavy (~50 kDa) and light (~25 kDa) chains.
    Figure Legend Snippet: Truncated LAP2 has multiple unique cytoplasmic interaction partners, including p62/SQSTM1. (A) Huh7 cells were transfected with GFP-tagged LAP2α or truncated LAP2 (1-99), followed by immunoprecipitation using an anti-GFP antibody; empty vector (GFP only) and GFP-tagged lamin A were included as controls. The immunoprecipitates and the input cell lysates were visualized by silver staining. (B) Huh7 cells were transfected with GFP alone, GFP-tagged LAP2α, or truncated LAP2 (1-99), followed by immunoprecipitation of LAP2 using anti-GFP antibody. Co-precipitated p62/SQSTM1 was visualized by immunoblotting. (C) Immunoprecipitated GFP and GFP-tagged proteins were visualized by Coomassie staining for the samples shown in panel B. M.W., apparent molecular weight; kDa, kilodaltons; IP, immunoprecipitates. Arrowheads indicate antibody heavy (~50 kDa) and light (~25 kDa) chains.

    Techniques Used: Transfection, Immunoprecipitation, Plasmid Preparation, Silver Staining, Staining, Molecular Weight

    Truncated LAP2 causes increased lipid accumulation in transfected cells. (A) Huh7 cells were transfected with DDK-tagged full-length (F.L.) LAP2α or truncated LAP2 (1-99) or empty vector, then treated with 500 μM oleic acid or vehicle (isopropanol) in serum-free medium overnight. After fixation, transfected LAP2 was visualized by indirect immunofluorescence using anti-FLAG antibody. Lipid droplets were stained with BODIPY 493/593 as described in Materials and Methods. Representative images are shown for each condition; scale bar, 20 μm. (B) Lipid accumulation was quantitated as described in Materials and Methods for > 10 high-power fields for each condition, and the data shown are representative of 3 independent experiments. Error bars represent standard error of the mean. Student’s t test was used to determine statistical significance at a threshold of P
    Figure Legend Snippet: Truncated LAP2 causes increased lipid accumulation in transfected cells. (A) Huh7 cells were transfected with DDK-tagged full-length (F.L.) LAP2α or truncated LAP2 (1-99) or empty vector, then treated with 500 μM oleic acid or vehicle (isopropanol) in serum-free medium overnight. After fixation, transfected LAP2 was visualized by indirect immunofluorescence using anti-FLAG antibody. Lipid droplets were stained with BODIPY 493/593 as described in Materials and Methods. Representative images are shown for each condition; scale bar, 20 μm. (B) Lipid accumulation was quantitated as described in Materials and Methods for > 10 high-power fields for each condition, and the data shown are representative of 3 independent experiments. Error bars represent standard error of the mean. Student’s t test was used to determine statistical significance at a threshold of P

    Techniques Used: Transfection, Plasmid Preparation, Immunofluorescence, Staining

    Truncated LAP2 causes altered lamin A/C distribution. (A) Huh7 cells were transfected with DDK-tagged full-length (F.L.) LAP2α or truncated LAP2 (1-99). After fixation, transfected LAP2 and endogenous lamin A/C were visualized by immunofluorescence using anti-FLAG and anti-lamin A/C antibodies, respectively. Representative high-magnification images are shown; nuclei of transfected cells with abnormal lamin A/C staining (punctate/globular) are highlighted by arrows. Scale bar, 20 μm. (B) Nuclear morphology and lamin A/C distribution in cells transfected with full-length LAP2α or truncated LAP2 (1-99) were scored in a blinded fashion from three independent experiments (3–6 fields/condition/experiment, > 85 total nuclei/condition). Error bars represent standard error of the mean. Student’s t test was used to determine statistical significance; ***, P
    Figure Legend Snippet: Truncated LAP2 causes altered lamin A/C distribution. (A) Huh7 cells were transfected with DDK-tagged full-length (F.L.) LAP2α or truncated LAP2 (1-99). After fixation, transfected LAP2 and endogenous lamin A/C were visualized by immunofluorescence using anti-FLAG and anti-lamin A/C antibodies, respectively. Representative high-magnification images are shown; nuclei of transfected cells with abnormal lamin A/C staining (punctate/globular) are highlighted by arrows. Scale bar, 20 μm. (B) Nuclear morphology and lamin A/C distribution in cells transfected with full-length LAP2α or truncated LAP2 (1-99) were scored in a blinded fashion from three independent experiments (3–6 fields/condition/experiment, > 85 total nuclei/condition). Error bars represent standard error of the mean. Student’s t test was used to determine statistical significance; ***, P

    Techniques Used: Transfection, Immunofluorescence, Staining

    28) Product Images from "Iodine-125 Seeds Inhibit Carcinogenesis of Hepatocellular Carcinoma Cells by Suppressing Epithelial-Mesenchymal Transition via TGF-β1/Smad Signaling"

    Article Title: Iodine-125 Seeds Inhibit Carcinogenesis of Hepatocellular Carcinoma Cells by Suppressing Epithelial-Mesenchymal Transition via TGF-β1/Smad Signaling

    Journal: Disease Markers

    doi: 10.1155/2022/9230647

    I-125 seed irradiation inhibits the TGF- β 1 signaling pathway. (a) RT-PCR assay demonstrated that the I-125 seed radiation downregulate the expression of TGF- β 1, Smad2, and Snail. (b). WB assay demonstrated that the I-125 seed radiation downregulate the expression of TGF- β 1, p-Smad2, and p-Smad3. (c) By immunofluorescence staining, the I-125 seed irradiation downregulates the expression of TGF- β 1 in PLC and Huh7 cells, respectively (data of the immunofluorescence for Smad2, p-Smad2, Smad3, and p-Smad3 not shown, bar: 20 μ m). (d) Relative protein expression of the genes in PLC. (e) Relative protein expression of the genes in Huh7. Data presented as the mean ± standard error of the mean ( n = 3). ∗ P
    Figure Legend Snippet: I-125 seed irradiation inhibits the TGF- β 1 signaling pathway. (a) RT-PCR assay demonstrated that the I-125 seed radiation downregulate the expression of TGF- β 1, Smad2, and Snail. (b). WB assay demonstrated that the I-125 seed radiation downregulate the expression of TGF- β 1, p-Smad2, and p-Smad3. (c) By immunofluorescence staining, the I-125 seed irradiation downregulates the expression of TGF- β 1 in PLC and Huh7 cells, respectively (data of the immunofluorescence for Smad2, p-Smad2, Smad3, and p-Smad3 not shown, bar: 20 μ m). (d) Relative protein expression of the genes in PLC. (e) Relative protein expression of the genes in Huh7. Data presented as the mean ± standard error of the mean ( n = 3). ∗ P

    Techniques Used: Irradiation, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Immunofluorescence, Staining, Planar Chromatography

    TGF- β 1 activator reverse I-125 seed irradiation inhibitory effects on the EMT and TGF- β 1 signaling pathway. TGF- β 1 activator reverses the inhibitory role in migration (a) and invasion (b) of PLC and Huh7. TGF- β 1 activator reverses the roles in the EMT and TGF- β 1 signaling pathway-related markers by WB assay (c) and immunofluorescence assay for PLC (d) and for Huh7 (e) and their relative IOD (integrated optical density) per cell were plotted, respectively (f). The average number of invasive cells was calculated by counting then number of cells in 5 fields per chamber. Data was expressed as mean ± SD ( n = 3). The difference was of statistical significance ( P
    Figure Legend Snippet: TGF- β 1 activator reverse I-125 seed irradiation inhibitory effects on the EMT and TGF- β 1 signaling pathway. TGF- β 1 activator reverses the inhibitory role in migration (a) and invasion (b) of PLC and Huh7. TGF- β 1 activator reverses the roles in the EMT and TGF- β 1 signaling pathway-related markers by WB assay (c) and immunofluorescence assay for PLC (d) and for Huh7 (e) and their relative IOD (integrated optical density) per cell were plotted, respectively (f). The average number of invasive cells was calculated by counting then number of cells in 5 fields per chamber. Data was expressed as mean ± SD ( n = 3). The difference was of statistical significance ( P

    Techniques Used: Irradiation, Migration, Planar Chromatography, Western Blot, Immunofluorescence

    I-125 seed irradiation suppresses cell proliferation, migration, and invasion of PLC and Huh7. (a) Cell viability of PLC and Huh7 cells was evaluated after 24 h of incubation with I-125 seed irradiation at varied doses (0, 2, 4, 6, and 8 Gy). Migration capabilities of PLC and Huh7 cells with the treatment of 0 and 4 Gy I-125 irradiation was assessed with wound healing assay (b) and migration assay (c). Invasive capabilities of PLC and Huh7 cells with the treatment of 0 and 4 Gy I-125 irradiation was assessed with Transwell assay (d). The average number of invasive cells was calculated by counting the number of cells in 5 fields per chamber. The expression of data as the mean ± standard error ( n = 3); ∗ P
    Figure Legend Snippet: I-125 seed irradiation suppresses cell proliferation, migration, and invasion of PLC and Huh7. (a) Cell viability of PLC and Huh7 cells was evaluated after 24 h of incubation with I-125 seed irradiation at varied doses (0, 2, 4, 6, and 8 Gy). Migration capabilities of PLC and Huh7 cells with the treatment of 0 and 4 Gy I-125 irradiation was assessed with wound healing assay (b) and migration assay (c). Invasive capabilities of PLC and Huh7 cells with the treatment of 0 and 4 Gy I-125 irradiation was assessed with Transwell assay (d). The average number of invasive cells was calculated by counting the number of cells in 5 fields per chamber. The expression of data as the mean ± standard error ( n = 3); ∗ P

    Techniques Used: Irradiation, Migration, Planar Chromatography, Incubation, Wound Healing Assay, Transwell Assay, Expressing

    Graphical abstract: TGF- β signaling mediates EMT. (a) TGF- β is an important player in the activation of EMT, which is characterized by downregulation of epithelial markers and upregulation of mesenchymal markers. TGF- β via either Smad or non-Smad signaling can promote EMT. The polyhedral fibroblasts were replaced by typical cobblestone like epithelioid cells when EMT takes place. The certain radiation of gamma-ray dose from I-125 seed increased production of reactive oxygen species (ROS) in almost tested cells, which shows downregulation of TGF- β 1, Smad2, Snail, N-cadherin, and vimentin in our tested cells by RT-PCR and WB assay. Inhibition of the TGF- β 1/Smad signaling pathway causes the inhibition of invasion and metastasis. The diagram has been partly adapted from the concepts of ref. 34 , 35 .
    Figure Legend Snippet: Graphical abstract: TGF- β signaling mediates EMT. (a) TGF- β is an important player in the activation of EMT, which is characterized by downregulation of epithelial markers and upregulation of mesenchymal markers. TGF- β via either Smad or non-Smad signaling can promote EMT. The polyhedral fibroblasts were replaced by typical cobblestone like epithelioid cells when EMT takes place. The certain radiation of gamma-ray dose from I-125 seed increased production of reactive oxygen species (ROS) in almost tested cells, which shows downregulation of TGF- β 1, Smad2, Snail, N-cadherin, and vimentin in our tested cells by RT-PCR and WB assay. Inhibition of the TGF- β 1/Smad signaling pathway causes the inhibition of invasion and metastasis. The diagram has been partly adapted from the concepts of ref. 34 , 35 .

    Techniques Used: Activation Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Inhibition

    I-125 seed irradiation inhibits EMT of PLC and Huh7 cells. (a) Morphological changes of PLC (upper panel) and Huh7 (lower panel) cells after irradiation (red arrow indicates the EMT site). (b) By immunofluorescence staining, I-125 seed irradiation upregulated the expression of E-cadherin in PLC (left) and Huh7 (right) cells. (c) RT-PCR assay demonstrates that I-125 seed radiation upregulated the expression of E-cadherin but downregulate that of N-cadherin and vimentin. (d, e) WB assay indicated that I-125 seed radiation increase the expression of E-cadherin and weak that of N-cadherin and vimentin. The expression of data as the mean ± standard error of the mean ( n = 3). ∗ P
    Figure Legend Snippet: I-125 seed irradiation inhibits EMT of PLC and Huh7 cells. (a) Morphological changes of PLC (upper panel) and Huh7 (lower panel) cells after irradiation (red arrow indicates the EMT site). (b) By immunofluorescence staining, I-125 seed irradiation upregulated the expression of E-cadherin in PLC (left) and Huh7 (right) cells. (c) RT-PCR assay demonstrates that I-125 seed radiation upregulated the expression of E-cadherin but downregulate that of N-cadherin and vimentin. (d, e) WB assay indicated that I-125 seed radiation increase the expression of E-cadherin and weak that of N-cadherin and vimentin. The expression of data as the mean ± standard error of the mean ( n = 3). ∗ P

    Techniques Used: Irradiation, Planar Chromatography, Immunofluorescence, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

    I-125 seed radiation suppressed the growth of PLC cells xenografts in mice. (a) Growth curves presented the volumes of xenograft tumor treated with I-125 seed radiation. (b) Images of the harvested tumors. (c) Weight of harvested tumors was measured 28 days after treatment. H E staining (d) and immunohistochemistry (e). The positive expression of TGF- β 1 in control and I-125 was quantitatively analyzed (f). The expression of data as mean ± SD ( n = 5). ∗∗ P
    Figure Legend Snippet: I-125 seed radiation suppressed the growth of PLC cells xenografts in mice. (a) Growth curves presented the volumes of xenograft tumor treated with I-125 seed radiation. (b) Images of the harvested tumors. (c) Weight of harvested tumors was measured 28 days after treatment. H E staining (d) and immunohistochemistry (e). The positive expression of TGF- β 1 in control and I-125 was quantitatively analyzed (f). The expression of data as mean ± SD ( n = 5). ∗∗ P

    Techniques Used: Planar Chromatography, Mouse Assay, Staining, Immunohistochemistry, Expressing

    29) Product Images from "RIG-I-Mediated STING Upregulation Restricts Herpes Simplex Virus 1 Infection"

    Article Title: RIG-I-Mediated STING Upregulation Restricts Herpes Simplex Virus 1 Infection

    Journal: Journal of Virology

    doi: 10.1128/JVI.00748-16

    Expression of STING is upregulated upon RIG-I signaling. A549 cells (A, B), IHHs, PC3 cells, U87 cells, Huh7.5 cells, or Huh7 cells (C), Ddx58 +/+ or Ddx58 −/− MEFs (D), and shctl- and shMAVS-transfected A549 cells (F) were uninfected or
    Figure Legend Snippet: Expression of STING is upregulated upon RIG-I signaling. A549 cells (A, B), IHHs, PC3 cells, U87 cells, Huh7.5 cells, or Huh7 cells (C), Ddx58 +/+ or Ddx58 −/− MEFs (D), and shctl- and shMAVS-transfected A549 cells (F) were uninfected or

    Techniques Used: Expressing, Transfection

    30) Product Images from "A Versatile Reporter System To Monitor Virus-Infected Cells and Its Application to Dengue Virus and SARS-CoV-2"

    Article Title: A Versatile Reporter System To Monitor Virus-Infected Cells and Its Application to Dengue Virus and SARS-CoV-2

    Journal: Journal of Virology

    doi: 10.1128/JVI.01715-20

    Evaluation of DENV reporter constructs. (A) Huh7 cells were transduced with lentiviruses encoding the different DENV GFP-based reporter constructs 1 to 8 ( Table 1 ) at an MOI of 5. Cells were fixed at 72 h postransduction and the subcellular distribution of GFP was analyzed by confocal microscopy. Scale bar = 100 μm. (B) Quantification of images acquired as in panel A. The percentage of cells showing nuclear or cytosolic GFP localization is shown. For each construct, more than 70 cells were counted. (C) Huh7 cells were transduced as above for 24 h before being infected with DENV2 at an MOI of 5. Cells were fixed at 48 hpi and NS3 was stained by immunofluorescence. Samples were analyzed by confocal microscopy. Magenta, DENV NS3 protein; green, reporter GFP signal. Scale bar = 100 μm. (D) Quantification of images as in panel C. The percentages of cells positive for NS3 and positive for both nuclear GFP and NS3 signals were quantified. For each construct, more than 60 cells were counted. (E) Cells expressing the reporter constructs 1 to 8 or an empty plasmid (empty) were infected with DENV2 (MOI = 5). At 48 hpi, cells were lysed and 10 μg of total protein for each sample was resolved by SDS-PAGE. NS3 and GFP were detected with a specific antibody. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) served as a loading control. Black arrowheads, uncleaved reporter; red arrowheads, reporter cleavage products. (F) Huh7-derived cells expressing reporter construct 1 (Huh7-RC) were infected with DENV serotypes 1 to 4 at an MOI of 5. Cells were fixed at 48 hpi and stained for double-stranded RNA (dsRNA) by IF. GFP and dsRNA signals were detected by fluorescence microscopy. For each serotype, more than 200 cells from at least two fields of view in duplicate were screened. Scale bar = 50 μm.
    Figure Legend Snippet: Evaluation of DENV reporter constructs. (A) Huh7 cells were transduced with lentiviruses encoding the different DENV GFP-based reporter constructs 1 to 8 ( Table 1 ) at an MOI of 5. Cells were fixed at 72 h postransduction and the subcellular distribution of GFP was analyzed by confocal microscopy. Scale bar = 100 μm. (B) Quantification of images acquired as in panel A. The percentage of cells showing nuclear or cytosolic GFP localization is shown. For each construct, more than 70 cells were counted. (C) Huh7 cells were transduced as above for 24 h before being infected with DENV2 at an MOI of 5. Cells were fixed at 48 hpi and NS3 was stained by immunofluorescence. Samples were analyzed by confocal microscopy. Magenta, DENV NS3 protein; green, reporter GFP signal. Scale bar = 100 μm. (D) Quantification of images as in panel C. The percentages of cells positive for NS3 and positive for both nuclear GFP and NS3 signals were quantified. For each construct, more than 60 cells were counted. (E) Cells expressing the reporter constructs 1 to 8 or an empty plasmid (empty) were infected with DENV2 (MOI = 5). At 48 hpi, cells were lysed and 10 μg of total protein for each sample was resolved by SDS-PAGE. NS3 and GFP were detected with a specific antibody. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) served as a loading control. Black arrowheads, uncleaved reporter; red arrowheads, reporter cleavage products. (F) Huh7-derived cells expressing reporter construct 1 (Huh7-RC) were infected with DENV serotypes 1 to 4 at an MOI of 5. Cells were fixed at 48 hpi and stained for double-stranded RNA (dsRNA) by IF. GFP and dsRNA signals were detected by fluorescence microscopy. For each serotype, more than 200 cells from at least two fields of view in duplicate were screened. Scale bar = 50 μm.

    Techniques Used: Construct, Transduction, Confocal Microscopy, Infection, Staining, Immunofluorescence, Expressing, Plasmid Preparation, SDS Page, Derivative Assay, Fluorescence, Microscopy

    Time-course experiments using the DENV reporter system in infection and transfection settings. (A) Huh7 cells stably expressing the reporter construct 1 were infected with DENV2 WT, the reporter virus DENV-faR (MOI = 5), or left uninfected. Cells were fixed at the indicated time points postinfection (hpi) and signals of the reporter virus (magenta) and the GFP-based reporter construct (green) were detected with a wide-field fluorescence microscope. Scale bar = 100 μm. (B) Quantification of images acquired as in panel A. The percentages of cells positive for nuclear GFP signal (Nuclear-GFP), DENV-faR reporter virus (Nuclear-faR), and double positive for both nuclear GFP and faR reporter signals (orange) were quantified. For each time point, more than 50 cells were analyzed. Values were normalized by setting the total number of cells counted using DAPI staining to 100%. (C) Huh7 cells stably expressing the reporter construct 1 were infected with DENV2 (MOI = 5). Cells were fixed at the indicated hpi and viral double-stranded RNA (dsRNA) was stained by IF. dsRNA (magenta) and GFP (green) signals were detected by confocal microscopy. Scale bar = 50 μm. (D) Quantification of cells using acquired images from the experiment shown in panel C. Percentages of cells positive for the nuclear GFP signal (green) and dsRNA (magenta) were quantified. For each time point, more than 250 cells were counted. Values were normalized by setting the total number of cells as determined by DAPI staining to 100%. (E) Experimental set-up to monitor GFP-reporter activation in pIRO-D transfected live cells. (F) Lunet-T7-RC cells stably expressing the T7 RNA polymerase and the reporter construct 1 were mock- or pIRO-D-transfected. Four hours after transfection, the medium was changed and live cell imaging was performed for 10 h using a confocal spinning disc microscope. Images of representative fields of view and the indicated time points are shown. Scale bar = 20 μm. See also Movie S1 in the supplemental material.
    Figure Legend Snippet: Time-course experiments using the DENV reporter system in infection and transfection settings. (A) Huh7 cells stably expressing the reporter construct 1 were infected with DENV2 WT, the reporter virus DENV-faR (MOI = 5), or left uninfected. Cells were fixed at the indicated time points postinfection (hpi) and signals of the reporter virus (magenta) and the GFP-based reporter construct (green) were detected with a wide-field fluorescence microscope. Scale bar = 100 μm. (B) Quantification of images acquired as in panel A. The percentages of cells positive for nuclear GFP signal (Nuclear-GFP), DENV-faR reporter virus (Nuclear-faR), and double positive for both nuclear GFP and faR reporter signals (orange) were quantified. For each time point, more than 50 cells were analyzed. Values were normalized by setting the total number of cells counted using DAPI staining to 100%. (C) Huh7 cells stably expressing the reporter construct 1 were infected with DENV2 (MOI = 5). Cells were fixed at the indicated hpi and viral double-stranded RNA (dsRNA) was stained by IF. dsRNA (magenta) and GFP (green) signals were detected by confocal microscopy. Scale bar = 50 μm. (D) Quantification of cells using acquired images from the experiment shown in panel C. Percentages of cells positive for the nuclear GFP signal (green) and dsRNA (magenta) were quantified. For each time point, more than 250 cells were counted. Values were normalized by setting the total number of cells as determined by DAPI staining to 100%. (E) Experimental set-up to monitor GFP-reporter activation in pIRO-D transfected live cells. (F) Lunet-T7-RC cells stably expressing the T7 RNA polymerase and the reporter construct 1 were mock- or pIRO-D-transfected. Four hours after transfection, the medium was changed and live cell imaging was performed for 10 h using a confocal spinning disc microscope. Images of representative fields of view and the indicated time points are shown. Scale bar = 20 μm. See also Movie S1 in the supplemental material.

    Techniques Used: Infection, Transfection, Stable Transfection, Expressing, Construct, Fluorescence, Microscopy, Staining, Confocal Microscopy, Activation Assay, Live Cell Imaging

    31) Product Images from "Cell-Type-Dependent Regulation of mTORC1 by REDD1 and the Tumor Suppressors TSC1/TSC2 and LKB1 in Response to Hypoxia ▿"

    Article Title: Cell-Type-Dependent Regulation of mTORC1 by REDD1 and the Tumor Suppressors TSC1/TSC2 and LKB1 in Response to Hypoxia ▿

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01393-10

    AMPK and raptor phosphorylation are essential for mTORC1 inhibition by hypoxia in liver cells. (A) Western blot of Huh7 cells transfected with the indicated siRNAs (or a scrambled control [Sc]) and exposed to hypoxia for the indicated number of hours. (B) Western blot of Huh7 cells pretreated (+) with compound C (Comp C) and exposed to hypoxia for the indicated number of hours. (C) Western blot of Huh7 cells stably transduced with AMPK dominant-negative (DN) mutants (T172A [TA] or K45R [KR] mutant) or an empty vector (EV) control and exposed to hypoxia (1% O 2 ) (H) or 2-deoxyglucose (2DG) (normoxia [N]). (D) Western blots of Huh7 cells in which endogenous raptor has been replaced by a phosphorylation-deficient raptor S722A/S792A (AA) or by a wild-type raptor control (WT).
    Figure Legend Snippet: AMPK and raptor phosphorylation are essential for mTORC1 inhibition by hypoxia in liver cells. (A) Western blot of Huh7 cells transfected with the indicated siRNAs (or a scrambled control [Sc]) and exposed to hypoxia for the indicated number of hours. (B) Western blot of Huh7 cells pretreated (+) with compound C (Comp C) and exposed to hypoxia for the indicated number of hours. (C) Western blot of Huh7 cells stably transduced with AMPK dominant-negative (DN) mutants (T172A [TA] or K45R [KR] mutant) or an empty vector (EV) control and exposed to hypoxia (1% O 2 ) (H) or 2-deoxyglucose (2DG) (normoxia [N]). (D) Western blots of Huh7 cells in which endogenous raptor has been replaced by a phosphorylation-deficient raptor S722A/S792A (AA) or by a wild-type raptor control (WT).

    Techniques Used: Inhibition, Western Blot, Transfection, Stable Transfection, Transduction, Dominant Negative Mutation, Mutagenesis, Plasmid Preparation

    32) Product Images from "Host Translation Shutoff Mediated by Non-structural Protein 2 is a Critical Factor in the Antiviral State Resistance of Venezuelan Equine Encephalitis Virus"

    Article Title: Host Translation Shutoff Mediated by Non-structural Protein 2 is a Critical Factor in the Antiviral State Resistance of Venezuelan Equine Encephalitis Virus

    Journal: Virology

    doi: 10.1016/j.virol.2016.06.005

    Individually expressed viral proteins block transcription and translation (A) Huh7 cells were transfected with indicated plasmids and lysates were collected at 18h post transfection. Western blots for HA-tag were performed as described in Materials and Methods. (B and C) Huh7 cells were transfected with plasmids coding for indicated viral proteins and labeled with 100μCi/ml of [ 35 S] Cys/Met for 2h at 8-24h post transfection. Lysates were collected and resolved on SDS-PAGE gels and visualized as described in Materials and Methods. (B) Representative image of nsP and capsid induced shutoff compared to GFP control. (C) Densitometry was performed to quantify the extent of shutoff following transfection of indicated plasmids. ****, P
    Figure Legend Snippet: Individually expressed viral proteins block transcription and translation (A) Huh7 cells were transfected with indicated plasmids and lysates were collected at 18h post transfection. Western blots for HA-tag were performed as described in Materials and Methods. (B and C) Huh7 cells were transfected with plasmids coding for indicated viral proteins and labeled with 100μCi/ml of [ 35 S] Cys/Met for 2h at 8-24h post transfection. Lysates were collected and resolved on SDS-PAGE gels and visualized as described in Materials and Methods. (B) Representative image of nsP and capsid induced shutoff compared to GFP control. (C) Densitometry was performed to quantify the extent of shutoff following transfection of indicated plasmids. ****, P

    Techniques Used: Blocking Assay, Transfection, Western Blot, Labeling, SDS Page

    33) Product Images from "The cellular NMD pathway restricts Zika virus infection and is targeted by the viral capsid protein"

    Article Title: The cellular NMD pathway restricts Zika virus infection and is targeted by the viral capsid protein

    Journal: bioRxiv

    doi: 10.1101/290296

    UPF1 is not transcriptionally downregulated during ZIKV infection or following ZIKV capsid overexpression. (a) UPF1 transcript levels from Huh7 cells mock-infected or infected with ZIKV strain PRVABC59 at an MOI of 0.1 or 1 and harvested at 48 hpi. Data are represented as mean ± s.e.m. P values were calculated by unpaired Student’s t -test. ns, not significant. n= 3 independent experiments. (b) UPF1 transcript levels from HEK293T cells transfected with vector or Strep-tagged ZIKV capsid (H/PF/2013, Asian lineage) and harvested at 48 hpt. Data are represented as mean ± s.e.m. P values were calculated by unpaired Student’s t -test. ns, not significant. n= 3 independent experiments.
    Figure Legend Snippet: UPF1 is not transcriptionally downregulated during ZIKV infection or following ZIKV capsid overexpression. (a) UPF1 transcript levels from Huh7 cells mock-infected or infected with ZIKV strain PRVABC59 at an MOI of 0.1 or 1 and harvested at 48 hpi. Data are represented as mean ± s.e.m. P values were calculated by unpaired Student’s t -test. ns, not significant. n= 3 independent experiments. (b) UPF1 transcript levels from HEK293T cells transfected with vector or Strep-tagged ZIKV capsid (H/PF/2013, Asian lineage) and harvested at 48 hpt. Data are represented as mean ± s.e.m. P values were calculated by unpaired Student’s t -test. ns, not significant. n= 3 independent experiments.

    Techniques Used: Infection, Over Expression, Transfection, Plasmid Preparation

    ZIKV capsid degrades UPF1, the master regulator of NMD, via a proteasome-dependent mechanism. (a) Western blot analysis of UPF1 levels in subcellular fractionated HEK293T cells transfected with vector or Flag-tagged ZIKV capsid (H/PF/2013, Asian lineage) for 48 h. GAPDH was used as a cytoplasmic marker and SP1 as a nuclear marker to ensure optimal fractionation. Densitometric analyses were performed using ImageJ to quantify relative band intensities. Data are represented as mean ± s.e.m. P values were calculated by unpaired Student’s t -test. ** P ≤ 0.01; ns, not significant. n= 3 independent experiments. (b) Western blot analysis of nuclear UPF1 levels in fractionated HEK293T cells transfected with vector or Flag-tagged ZIKV capsid for 48 h. Cells were treated with DMSO or increasing concentrations of the proteasome inhibitor bortezomib for 24 h before harvest. Densitometric analyses were performed using ImageJ to quantify relative band intensities. Data are represented as mean ± s.e.m. P values were calculated by one-way ANOVA with multiple comparisons. * P ≤ 0.05; ns, not significant. n= 3 independent experiments. (c) Representative 3D confocal microscopy images of the nuclei of Huh7-Lunet cells transfected with Strep-tagged ZIKV capsid. Cells were treated at 24 hpt with DMSO or 10 nM bortezomib and processed for immunostaining at 48 hpt with antibodies against Strep-tag (turquoise) and endogenous UPF1 (purple). DAPI (blue) was used to stain and define the nuclei. Each channel was reconstructed digitally for visualization of the 3D colocalization. The thresholded Mander’s correlation coefficients were determined and P value was calculated by unpaired Student’s t -test. ** P ≤ 0.01. n = 8 cells per condition. Scale bar represents 3 μm.
    Figure Legend Snippet: ZIKV capsid degrades UPF1, the master regulator of NMD, via a proteasome-dependent mechanism. (a) Western blot analysis of UPF1 levels in subcellular fractionated HEK293T cells transfected with vector or Flag-tagged ZIKV capsid (H/PF/2013, Asian lineage) for 48 h. GAPDH was used as a cytoplasmic marker and SP1 as a nuclear marker to ensure optimal fractionation. Densitometric analyses were performed using ImageJ to quantify relative band intensities. Data are represented as mean ± s.e.m. P values were calculated by unpaired Student’s t -test. ** P ≤ 0.01; ns, not significant. n= 3 independent experiments. (b) Western blot analysis of nuclear UPF1 levels in fractionated HEK293T cells transfected with vector or Flag-tagged ZIKV capsid for 48 h. Cells were treated with DMSO or increasing concentrations of the proteasome inhibitor bortezomib for 24 h before harvest. Densitometric analyses were performed using ImageJ to quantify relative band intensities. Data are represented as mean ± s.e.m. P values were calculated by one-way ANOVA with multiple comparisons. * P ≤ 0.05; ns, not significant. n= 3 independent experiments. (c) Representative 3D confocal microscopy images of the nuclei of Huh7-Lunet cells transfected with Strep-tagged ZIKV capsid. Cells were treated at 24 hpt with DMSO or 10 nM bortezomib and processed for immunostaining at 48 hpt with antibodies against Strep-tag (turquoise) and endogenous UPF1 (purple). DAPI (blue) was used to stain and define the nuclei. Each channel was reconstructed digitally for visualization of the 3D colocalization. The thresholded Mander’s correlation coefficients were determined and P value was calculated by unpaired Student’s t -test. ** P ≤ 0.01. n = 8 cells per condition. Scale bar represents 3 μm.

    Techniques Used: Western Blot, Transfection, Plasmid Preparation, Marker, Fractionation, Confocal Microscopy, Immunostaining, Strep-tag, Staining

    ZIKV infection disrupts the NMD pathway. (a) Transcript levels of NMD substrates and housekeeping genes from Huh7 cells or NPCs mock-infected or infected with ZIKV strain P6-740 or the contemporary clinical isolate PRVABC59. Cells were infected at a multiplicity of infection (MOI) of 0.1 or 1 and harvested at 48 hpi. Data are represented as mean ± s.e.m. P values were calculated by unpaired Student’s t -test. * P ≤ 0.05; ** P ≤ 0.01; ns, not significant. n= 3 independent experiments. (b) Venn diagram showing overlap of significantly upregulated genes associated with ZIKV infection of NPCs and UPF1 knockdown in HeLa cells. RNA-Seq analyses of mock-infected or ZIKV-infected NPCs harvested at 56 hpi and control siRNA-treated or UPF1 siRNA-treated HeLa TO cells harvested at 72 h post-transfection (hpt). The GeneProf hypergeometric probability calculator was then used to generate a hypergeometric P value. **** P ≤ 0.0001. (c) Transcript levels of housekeeping genes and select genes involved in cell cycle growth arrest and apoptosis that were identified in (b). Huh7 cells were mock-infected or infected with ZIKV PRVABC59 at an MOI of 0.1 or 1 and harvested at 48 hpi. Data are represented as mean ± s.e.m. P values were calculated by unpaired Student’s t -test. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; ns, not significant. n= 3 independent experiments.
    Figure Legend Snippet: ZIKV infection disrupts the NMD pathway. (a) Transcript levels of NMD substrates and housekeeping genes from Huh7 cells or NPCs mock-infected or infected with ZIKV strain P6-740 or the contemporary clinical isolate PRVABC59. Cells were infected at a multiplicity of infection (MOI) of 0.1 or 1 and harvested at 48 hpi. Data are represented as mean ± s.e.m. P values were calculated by unpaired Student’s t -test. * P ≤ 0.05; ** P ≤ 0.01; ns, not significant. n= 3 independent experiments. (b) Venn diagram showing overlap of significantly upregulated genes associated with ZIKV infection of NPCs and UPF1 knockdown in HeLa cells. RNA-Seq analyses of mock-infected or ZIKV-infected NPCs harvested at 56 hpi and control siRNA-treated or UPF1 siRNA-treated HeLa TO cells harvested at 72 h post-transfection (hpt). The GeneProf hypergeometric probability calculator was then used to generate a hypergeometric P value. **** P ≤ 0.0001. (c) Transcript levels of housekeeping genes and select genes involved in cell cycle growth arrest and apoptosis that were identified in (b). Huh7 cells were mock-infected or infected with ZIKV PRVABC59 at an MOI of 0.1 or 1 and harvested at 48 hpi. Data are represented as mean ± s.e.m. P values were calculated by unpaired Student’s t -test. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; ns, not significant. n= 3 independent experiments.

    Techniques Used: Infection, RNA Sequencing Assay, Transfection

    ZIKV capsid colocalizes with endogenous UPF1. Representative 3D confocal microscopy images of Huh7-Lunet cells transfected with vector or Strep-tagged ZIKV capsid. Cells were processed for immunostaining at 48 hpt and probed with antibodies against Strep-tag (turquoise) and endogenous UPF1 (purple). DAPI (blue) was used to stain the nuclei. Each channel was reconstructed digitally for visualization of the 3D colocalization. The thresholded Mander’s correlation coefficient for ZIKV capsid was 0.57 (n = 17), indicating that approximately 57% of the voxels positive for capsid fluorescence were also positive for UPF1 fluorescence. Scale bar represents 5 μm.
    Figure Legend Snippet: ZIKV capsid colocalizes with endogenous UPF1. Representative 3D confocal microscopy images of Huh7-Lunet cells transfected with vector or Strep-tagged ZIKV capsid. Cells were processed for immunostaining at 48 hpt and probed with antibodies against Strep-tag (turquoise) and endogenous UPF1 (purple). DAPI (blue) was used to stain the nuclei. Each channel was reconstructed digitally for visualization of the 3D colocalization. The thresholded Mander’s correlation coefficient for ZIKV capsid was 0.57 (n = 17), indicating that approximately 57% of the voxels positive for capsid fluorescence were also positive for UPF1 fluorescence. Scale bar represents 5 μm.

    Techniques Used: Confocal Microscopy, Transfection, Plasmid Preparation, Immunostaining, Strep-tag, Staining, Fluorescence

    34) Product Images from "The mRNA cap 2’O methyltransferase CMTR1 regulates the expression of certain interferon-stimulated genes"

    Article Title: The mRNA cap 2’O methyltransferase CMTR1 regulates the expression of certain interferon-stimulated genes

    Journal: bioRxiv

    doi: 10.1101/2020.03.05.980045

    CMTR1 does not regulate the nuclear export, RNA stability, or polysome association of specific ISGs. (A) The relative percent of ISGs or MALAT1 (nuclear fractionation control) in nuclear and cytoplasmic fractions of siRNA and IFN-β-treated Huh7 cells, as analyzed by RT-qPCR. (B) RNA stability, as measured by the percent of transcript remaining in siRNA and IFN-β-treated Huh7 cells at the indicated times following actinomycin D (10 µg/mL) treatment. (C) (Top) A representative plot of the relative absorbance values of fractions isolated from extracts of siRNA and IFN-β-treated Huh7 cells following centrifugation over 15-50% sucrose gradients. (Bottom) RNA from each fraction was separated on an agarose gel and visualized with ethidium bromide. Ribosomal RNA bands (18S, 12S, and 5S) are indicated. (D) RT-qPCR analysis of ISGs from polysome profiling of extracts from (C). Data are presented as the percent of total mRNA in each fraction. All IFN-β treatments were performed at 50 U/mL for 6 h. Values are the mean ± SEM of 3 biological replicates.
    Figure Legend Snippet: CMTR1 does not regulate the nuclear export, RNA stability, or polysome association of specific ISGs. (A) The relative percent of ISGs or MALAT1 (nuclear fractionation control) in nuclear and cytoplasmic fractions of siRNA and IFN-β-treated Huh7 cells, as analyzed by RT-qPCR. (B) RNA stability, as measured by the percent of transcript remaining in siRNA and IFN-β-treated Huh7 cells at the indicated times following actinomycin D (10 µg/mL) treatment. (C) (Top) A representative plot of the relative absorbance values of fractions isolated from extracts of siRNA and IFN-β-treated Huh7 cells following centrifugation over 15-50% sucrose gradients. (Bottom) RNA from each fraction was separated on an agarose gel and visualized with ethidium bromide. Ribosomal RNA bands (18S, 12S, and 5S) are indicated. (D) RT-qPCR analysis of ISGs from polysome profiling of extracts from (C). Data are presented as the percent of total mRNA in each fraction. All IFN-β treatments were performed at 50 U/mL for 6 h. Values are the mean ± SEM of 3 biological replicates.

    Techniques Used: Fractionation, Quantitative RT-PCR, Isolation, Centrifugation, Agarose Gel Electrophoresis

    CMTR1 inhibits IFIT1-mediated translational regulation of specific ISGs via their 5’ UTRs. (A) Representative immunoblot of extracts of IFN-β-treated Huh7 cells transduced with the indicated shRNA (non-targeting control (NT) and IFIT1) and then transfected with siCTRL or siCMTR1. (B) Quantification of immunoblots from (A). The values display the relative protein levels of the indicated ISG normalized to the total protein, which were then set to 1 for each set of siCTRL samples. They represent the mean ± SEM of 4 biological replicates. (C) Representative immunoblot of FLAG-immunoprecipitated (IP) or input fractions of IFN-β-treated Huh7 cells transfected with Vector or FLAG-IFIT1, as well as siCTRL or siCMTR1. (D) RT-qPCR analysis of enriched transcripts relative to input by anti-FLAG IP in siRNA and IFN-β-treated Huh7 cells. Fold enrichment values for each transcript are graphed relative to siCTRL and are the mean ± SEM of 3 biological replicates. (E) Secreted Gaussia luciferase units of a reporter whose expression is driven by the 5’ UTR of the indicated ISGs and contains either Cap 0 or Cap 1 in IFN-β-treated Huh7 cells transduced with shNT or shIFIT1. Fold change values for the indicated reporters are shown, with the shNT cells transfected with the indicated Cap 0 construct set to 1 for each experiment, and graphed as the mean ± SEM of 3 biological replicates. ( F ) Following IFN-β-signaling, IFIT1 and IFIT3 are induced, and they can inhibit the translation of Cap 0 mRNAs. However, CMTR1 induction by IFN-β can ensure that the mRNA of ISGs is modified by Cap 1, ensuring ISG protein expression and the antiviral response. For (B) and (D), ** p
    Figure Legend Snippet: CMTR1 inhibits IFIT1-mediated translational regulation of specific ISGs via their 5’ UTRs. (A) Representative immunoblot of extracts of IFN-β-treated Huh7 cells transduced with the indicated shRNA (non-targeting control (NT) and IFIT1) and then transfected with siCTRL or siCMTR1. (B) Quantification of immunoblots from (A). The values display the relative protein levels of the indicated ISG normalized to the total protein, which were then set to 1 for each set of siCTRL samples. They represent the mean ± SEM of 4 biological replicates. (C) Representative immunoblot of FLAG-immunoprecipitated (IP) or input fractions of IFN-β-treated Huh7 cells transfected with Vector or FLAG-IFIT1, as well as siCTRL or siCMTR1. (D) RT-qPCR analysis of enriched transcripts relative to input by anti-FLAG IP in siRNA and IFN-β-treated Huh7 cells. Fold enrichment values for each transcript are graphed relative to siCTRL and are the mean ± SEM of 3 biological replicates. (E) Secreted Gaussia luciferase units of a reporter whose expression is driven by the 5’ UTR of the indicated ISGs and contains either Cap 0 or Cap 1 in IFN-β-treated Huh7 cells transduced with shNT or shIFIT1. Fold change values for the indicated reporters are shown, with the shNT cells transfected with the indicated Cap 0 construct set to 1 for each experiment, and graphed as the mean ± SEM of 3 biological replicates. ( F ) Following IFN-β-signaling, IFIT1 and IFIT3 are induced, and they can inhibit the translation of Cap 0 mRNAs. However, CMTR1 induction by IFN-β can ensure that the mRNA of ISGs is modified by Cap 1, ensuring ISG protein expression and the antiviral response. For (B) and (D), ** p

    Techniques Used: Transduction, shRNA, Transfection, Western Blot, Immunoprecipitation, Plasmid Preparation, Quantitative RT-PCR, Luciferase, Expressing, Construct, Modification

    CMTR1 promotes the protein expression of specific ISGs. (A) Schematic of 5’ cap structure, with m 7 G (Cap 0) and 2’O methylation of the first transcribed nucleotide catalyzed by CMTR1 (Cap 1) (B) Fold change of CMTR1 treated with the indicated stimuli (type I, type II, or type III IFNs; viruses (human rhinovirus 16, Newcastle disease virus, human cytomegalovirus strain TB40E, influenza A virus, yellow fever virus 17D); pathogen-associated molecular patterns (PAMP: lipopolysaccharide or poly (I:C)) relative to untreated samples. Data were obtained from EMBL-EBI Expression Atlas RNA-seq database ( 34 ). (C) RT-qPCR analysis of CMTR1 relative to GAPDH in either Huh7 or THP-1 cells following IFN-β treatment, as compared to levels in untreated cells. (D) RT-qPCR analysis of CMTR1, ISG15 , and IFIT1 relative to GAPDH in mock-treated Huh7 cells transfected with the indicated siRNAs. (E) Representative immunoblot of extracts from mock- or IFN-β-treated Huh7 cells transfected with the indicated siRNAs. (F) Quantification of the immunoblots from (E), normalized to total protein and graphed relative to siCTRL. (G) RT-qPCR analysis of ISGs relative to GAPDH in IFN-β-treated Huh7 cells transfected with the indicated siRNAs. Data are graphed as the fold change relative to mock-treated cells. All IFN-β treatments were performed at 50 U/mL for 6 h. Values are the mean ± SEM of 3 (C, D, G) or 4 (F) biological replicates. * p
    Figure Legend Snippet: CMTR1 promotes the protein expression of specific ISGs. (A) Schematic of 5’ cap structure, with m 7 G (Cap 0) and 2’O methylation of the first transcribed nucleotide catalyzed by CMTR1 (Cap 1) (B) Fold change of CMTR1 treated with the indicated stimuli (type I, type II, or type III IFNs; viruses (human rhinovirus 16, Newcastle disease virus, human cytomegalovirus strain TB40E, influenza A virus, yellow fever virus 17D); pathogen-associated molecular patterns (PAMP: lipopolysaccharide or poly (I:C)) relative to untreated samples. Data were obtained from EMBL-EBI Expression Atlas RNA-seq database ( 34 ). (C) RT-qPCR analysis of CMTR1 relative to GAPDH in either Huh7 or THP-1 cells following IFN-β treatment, as compared to levels in untreated cells. (D) RT-qPCR analysis of CMTR1, ISG15 , and IFIT1 relative to GAPDH in mock-treated Huh7 cells transfected with the indicated siRNAs. (E) Representative immunoblot of extracts from mock- or IFN-β-treated Huh7 cells transfected with the indicated siRNAs. (F) Quantification of the immunoblots from (E), normalized to total protein and graphed relative to siCTRL. (G) RT-qPCR analysis of ISGs relative to GAPDH in IFN-β-treated Huh7 cells transfected with the indicated siRNAs. Data are graphed as the fold change relative to mock-treated cells. All IFN-β treatments were performed at 50 U/mL for 6 h. Values are the mean ± SEM of 3 (C, D, G) or 4 (F) biological replicates. * p

    Techniques Used: Expressing, Methylation, RNA Sequencing Assay, Quantitative RT-PCR, Transfection, Western Blot

    CMTR1 is required for the antiviral response. (A) Focus-forming assay of supernatants or (B) RT-qPCR analysis of viral RNA, relative to GAPDH , harvested from ZIKV- or DENV-infected Huh7 cells (48 hours post-infection (hpi); multiplicity of infection (MOI) 0.01) treated with the indicated siRNAs. (C) (Left) Representative fields of ZIKV- and DENV-infected Huh7 cells (48 hpi; MOI 0.01) treated with the indicated siRNAs and immunostained with anti-flaviviral E protein (green). Nuclei were stained with DAPI (blue). Scale bar: 100 μm. (Right) Quantification of the percentage of ZIKV- and DENV-infected Huh7 cells. (D) Quantification of the relative number of VSV-GFP-positive Huh7 WT and STAT1 KO cells after siRNA treatment and IFN-β pre-treatment (25 U/mL; 16 h) at 8 hpi and set to 100. For (C) and (D): ≥5000 cells were counted in each experiment per condition. Values are the mean ± SEM of 3 biological replicates. * p
    Figure Legend Snippet: CMTR1 is required for the antiviral response. (A) Focus-forming assay of supernatants or (B) RT-qPCR analysis of viral RNA, relative to GAPDH , harvested from ZIKV- or DENV-infected Huh7 cells (48 hours post-infection (hpi); multiplicity of infection (MOI) 0.01) treated with the indicated siRNAs. (C) (Left) Representative fields of ZIKV- and DENV-infected Huh7 cells (48 hpi; MOI 0.01) treated with the indicated siRNAs and immunostained with anti-flaviviral E protein (green). Nuclei were stained with DAPI (blue). Scale bar: 100 μm. (Right) Quantification of the percentage of ZIKV- and DENV-infected Huh7 cells. (D) Quantification of the relative number of VSV-GFP-positive Huh7 WT and STAT1 KO cells after siRNA treatment and IFN-β pre-treatment (25 U/mL; 16 h) at 8 hpi and set to 100. For (C) and (D): ≥5000 cells were counted in each experiment per condition. Values are the mean ± SEM of 3 biological replicates. * p

    Techniques Used: Focus Forming Assay, Quantitative RT-PCR, Infection, Staining

    35) Product Images from "Development of a reverse genetics system for Sosuga virus allows rapid screening of antiviral compounds"

    Article Title: Development of a reverse genetics system for Sosuga virus allows rapid screening of antiviral compounds

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0006326

    Optimization of an antiviral screening assay using a recombinant SOSV expressing ZsG. a. Schematic representation of the wild-type recombinant SOSV (rSOSV), and the reporter SOSV expressing ZsG (rSOSV/ZsG). The ZsG coding sequence was inserted immediately 5′ of the M coding sequence (antigenomic sense), separated from the viral protein by the P2A sequence from porcine teschovirus 1. This self-cleaving amino acid motif allows expression of both ZsG and M from a single mRNA generated by the parental SOSV M promoter and terminator sequences. b. Vero-E6 cells infected with either rSOSV or rSOSV/ZsG at 72 hpi showing formation of syncytia. In rSOSV/ZsG-infected cell monolayers, syncytia were associated with extensive ZsG expression (images taken at 4 × magnification). c. Growth curves for wild-type SOSV, rSOSV, and rSOSV/ZsG were performed in Vero-E6 cells infected at MOI 0.1. Titers (TCID 50 ) were determined at 24, 48, 72, 96, and 120 hpi. d. The optimized rSOSV/ZsG antiviral screening assay was validated using ribavirin. Huh7 cells were treated with a serial 2-fold dilution of ribavirin 1 h prior to infection at MOI 0.2. ZsG fluorescence (green) was measured at 72 hpi and normalized to levels in mock-treated cells (DMSO only). Cell viability (blue) was determined concurrently by measuring ATP content, with values normalized to mock-infected cells. Each point represents the mean of quadruplicate wells, with error bars showing standard deviation; graph representative of 3 independent experiments.
    Figure Legend Snippet: Optimization of an antiviral screening assay using a recombinant SOSV expressing ZsG. a. Schematic representation of the wild-type recombinant SOSV (rSOSV), and the reporter SOSV expressing ZsG (rSOSV/ZsG). The ZsG coding sequence was inserted immediately 5′ of the M coding sequence (antigenomic sense), separated from the viral protein by the P2A sequence from porcine teschovirus 1. This self-cleaving amino acid motif allows expression of both ZsG and M from a single mRNA generated by the parental SOSV M promoter and terminator sequences. b. Vero-E6 cells infected with either rSOSV or rSOSV/ZsG at 72 hpi showing formation of syncytia. In rSOSV/ZsG-infected cell monolayers, syncytia were associated with extensive ZsG expression (images taken at 4 × magnification). c. Growth curves for wild-type SOSV, rSOSV, and rSOSV/ZsG were performed in Vero-E6 cells infected at MOI 0.1. Titers (TCID 50 ) were determined at 24, 48, 72, 96, and 120 hpi. d. The optimized rSOSV/ZsG antiviral screening assay was validated using ribavirin. Huh7 cells were treated with a serial 2-fold dilution of ribavirin 1 h prior to infection at MOI 0.2. ZsG fluorescence (green) was measured at 72 hpi and normalized to levels in mock-treated cells (DMSO only). Cell viability (blue) was determined concurrently by measuring ATP content, with values normalized to mock-infected cells. Each point represents the mean of quadruplicate wells, with error bars showing standard deviation; graph representative of 3 independent experiments.

    Techniques Used: Screening Assay, Recombinant, Expressing, Sequencing, Generated, Infection, Fluorescence, Standard Deviation

    Design and optimization of the Sosuga virus minigenome screening assay. a. Genome schematic representing the design of the Sosuga virus (SOSV) minigenome segment. The minigenome contains the full-length SOSV 3′ and 5′ leader and trailer sequences, along with the gene start sequence for nucleoprotein (NP) and the gene end sequence for the viral polymerase (L), with the parental coding and intergenic regions replaced by the coding sequence of ZsGreen1 (ZsG). Transfected into cells in conjunction with plasmids expressing SOSV L, NP, and phosphoprotein (P), this minigenome allows for expression of quantifiable ZsG. b. The minigenome assay was optimized for Huh7 cells in a 96-well plate format using different ratios of the plasmids expressing L, NP, and P with a constant amount of minigenome plasmid. Cells were transfected with 75 ng minigenome plasmid and 75 ng total of plasmids expressing L, NP, and P, with ratios of each stated underneath the bars. Relative ZsG fluorescence over control reactions with no L was calculated at 48 and 72 h post transfection (hpt). c. Dose-response curve for the optimized SOSV minigenome assay against ribavirin. Cells were treated with a serial 2-fold dilution of ribavirin 1 hpt with the minigenome plasmids, and ZsG fluorescence was measured at 72 hpt. ZsG fluorescence (green) was normalized to mock-treated cells (DMSO only). Cell viability (blue) was determined concurrently by measuring ATP content, with values normalized to mock-transfected cells. Each point represents the mean of quadruplicate wells, with error bars showing standard deviation; graph is representative of 3 independent experiments.
    Figure Legend Snippet: Design and optimization of the Sosuga virus minigenome screening assay. a. Genome schematic representing the design of the Sosuga virus (SOSV) minigenome segment. The minigenome contains the full-length SOSV 3′ and 5′ leader and trailer sequences, along with the gene start sequence for nucleoprotein (NP) and the gene end sequence for the viral polymerase (L), with the parental coding and intergenic regions replaced by the coding sequence of ZsGreen1 (ZsG). Transfected into cells in conjunction with plasmids expressing SOSV L, NP, and phosphoprotein (P), this minigenome allows for expression of quantifiable ZsG. b. The minigenome assay was optimized for Huh7 cells in a 96-well plate format using different ratios of the plasmids expressing L, NP, and P with a constant amount of minigenome plasmid. Cells were transfected with 75 ng minigenome plasmid and 75 ng total of plasmids expressing L, NP, and P, with ratios of each stated underneath the bars. Relative ZsG fluorescence over control reactions with no L was calculated at 48 and 72 h post transfection (hpt). c. Dose-response curve for the optimized SOSV minigenome assay against ribavirin. Cells were treated with a serial 2-fold dilution of ribavirin 1 hpt with the minigenome plasmids, and ZsG fluorescence was measured at 72 hpt. ZsG fluorescence (green) was normalized to mock-treated cells (DMSO only). Cell viability (blue) was determined concurrently by measuring ATP content, with values normalized to mock-transfected cells. Each point represents the mean of quadruplicate wells, with error bars showing standard deviation; graph is representative of 3 independent experiments.

    Techniques Used: Screening Assay, Sequencing, Transfection, Expressing, Plasmid Preparation, Fluorescence, Standard Deviation

    Immunofluorescent imaging of rSOSV infected Huh7 cells treated with selected antiviral compounds. Huh7 cells were treated with serial dilutions of either 2′-deoxy-2′-fluorocytidine (2′-dFC), 6-azauridine, mycophenolic acid (MPA), or 09167 1 h prior to infection with rSOSV at MOI 0.2. At 48 hpi, cells were fixed and SOSV proteins (green) and cell nuclei (blue) visualized by immunofluorescence. Concentration of compound is shown in white text on each representative image (mM); control cells were treated with DMSO only. Images taken using a 20 × objective. White bar represents 100 μm.
    Figure Legend Snippet: Immunofluorescent imaging of rSOSV infected Huh7 cells treated with selected antiviral compounds. Huh7 cells were treated with serial dilutions of either 2′-deoxy-2′-fluorocytidine (2′-dFC), 6-azauridine, mycophenolic acid (MPA), or 09167 1 h prior to infection with rSOSV at MOI 0.2. At 48 hpi, cells were fixed and SOSV proteins (green) and cell nuclei (blue) visualized by immunofluorescence. Concentration of compound is shown in white text on each representative image (mM); control cells were treated with DMSO only. Images taken using a 20 × objective. White bar represents 100 μm.

    Techniques Used: Imaging, Infection, Immunofluorescence, Concentration Assay

    36) Product Images from "Hepatitis C Virus Protein Interaction Network Analysis Based on Hepatocellular Carcinoma"

    Article Title: Hepatitis C Virus Protein Interaction Network Analysis Based on Hepatocellular Carcinoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0153882

    NS5A promotes HCC cell proliferation and metastasis by activating AKT/GSK3β/β-catenin pathway. (A) The p-AKT, AKT, p-GSK3β, GSK3β and β-catenin protein expression levels were determined by western blot analysis from Huh7 cells (Blank), negative control cells (NC) or NS5A overexpression cells (NS5A). β-actin was used as internal control for normalization in western blotting. (B) The proliferation of NS5A overexpression cells were evaluated using the CCK-8 assay (at 0, 12, 24, and 36 h). (C) The migration of NS5A overexpression cells by using a scratch-healing assay. (D) The invasiveness of NS5A overexpression cells compared with the control cells by using a Matrigel-transwell assay system. Five fields of cells were counted for each well, and the mean number of cells per field was calculated. All data shown are from 2 independent experiments conducted in triplicate. * P
    Figure Legend Snippet: NS5A promotes HCC cell proliferation and metastasis by activating AKT/GSK3β/β-catenin pathway. (A) The p-AKT, AKT, p-GSK3β, GSK3β and β-catenin protein expression levels were determined by western blot analysis from Huh7 cells (Blank), negative control cells (NC) or NS5A overexpression cells (NS5A). β-actin was used as internal control for normalization in western blotting. (B) The proliferation of NS5A overexpression cells were evaluated using the CCK-8 assay (at 0, 12, 24, and 36 h). (C) The migration of NS5A overexpression cells by using a scratch-healing assay. (D) The invasiveness of NS5A overexpression cells compared with the control cells by using a Matrigel-transwell assay system. Five fields of cells were counted for each well, and the mean number of cells per field was calculated. All data shown are from 2 independent experiments conducted in triplicate. * P

    Techniques Used: Expressing, Western Blot, Negative Control, Over Expression, CCK-8 Assay, Migration, Transwell Assay

    37) Product Images from "Host Translation Shutoff Mediated by Non-structural Protein 2 is a Critical Factor in the Antiviral State Resistance of Venezuelan Equine Encephalitis Virus"

    Article Title: Host Translation Shutoff Mediated by Non-structural Protein 2 is a Critical Factor in the Antiviral State Resistance of Venezuelan Equine Encephalitis Virus

    Journal: Virology

    doi: 10.1016/j.virol.2016.06.005

    Individually expressed viral proteins block transcription and translation (A) Huh7 cells were transfected with indicated plasmids and lysates were collected at 18h post transfection. Western blots for HA-tag were performed as described in Materials and Methods. (B and C) Huh7 cells were transfected with plasmids coding for indicated viral proteins and labeled with 100μCi/ml of [ 35 S] Cys/Met for 2h at 8-24h post transfection. Lysates were collected and resolved on SDS-PAGE gels and visualized as described in Materials and Methods. (B) Representative image of nsP and capsid induced shutoff compared to GFP control. (C) Densitometry was performed to quantify the extent of shutoff following transfection of indicated plasmids. ****, P
    Figure Legend Snippet: Individually expressed viral proteins block transcription and translation (A) Huh7 cells were transfected with indicated plasmids and lysates were collected at 18h post transfection. Western blots for HA-tag were performed as described in Materials and Methods. (B and C) Huh7 cells were transfected with plasmids coding for indicated viral proteins and labeled with 100μCi/ml of [ 35 S] Cys/Met for 2h at 8-24h post transfection. Lysates were collected and resolved on SDS-PAGE gels and visualized as described in Materials and Methods. (B) Representative image of nsP and capsid induced shutoff compared to GFP control. (C) Densitometry was performed to quantify the extent of shutoff following transfection of indicated plasmids. ****, P

    Techniques Used: Blocking Assay, Transfection, Western Blot, Labeling, SDS Page

    38) Product Images from "A novel inhibitor of MDM2 oncogene blocks metastasis of hepatocellular carcinoma and overcomes chemoresistance"

    Article Title: A novel inhibitor of MDM2 oncogene blocks metastasis of hepatocellular carcinoma and overcomes chemoresistance

    Journal: Genes & Diseases

    doi: 10.1016/j.gendis.2019.06.001

    SP141 sensitizes hepatocellular carcinoma cells to sorafenib. (A) HepG2 and Huh7 cells with or without sgMDM2 were treated with SP141 (0.1 μM) and various concentrations (0, 0.5, 1, 2, 5 and 10 μM) of sorafenib for 72 h to evaluate the cell viability via MTT assays. (B) The cells with or without sgMDM2 were treated with SP141 (0.1 μM) and sorafenib (2 μM) for 24 h to measure the cell invasion via transwell invasion assays. (C) The cells with or without sgMDM2 were treated with SP141 (0.1 μM) and various concentrations of sorafenib for 24 h prior to analyses of the protein expression. All assays were performed in triplicate and repeated three times. (* P
    Figure Legend Snippet: SP141 sensitizes hepatocellular carcinoma cells to sorafenib. (A) HepG2 and Huh7 cells with or without sgMDM2 were treated with SP141 (0.1 μM) and various concentrations (0, 0.5, 1, 2, 5 and 10 μM) of sorafenib for 72 h to evaluate the cell viability via MTT assays. (B) The cells with or without sgMDM2 were treated with SP141 (0.1 μM) and sorafenib (2 μM) for 24 h to measure the cell invasion via transwell invasion assays. (C) The cells with or without sgMDM2 were treated with SP141 (0.1 μM) and various concentrations of sorafenib for 24 h prior to analyses of the protein expression. All assays were performed in triplicate and repeated three times. (* P

    Techniques Used: MTT Assay, Expressing

    SP141 induces MDM2 degradation in human HCC cells. (A) HepG2 and Huh7 cells were exposed to 1 μM of SP141 for 2 h, followed by cellular thermal shift assays. (B) HCC cells were exposed to various concentrations of SP141 for 24 h. The protein expression levels of MDM2, p53, and p21 were determined. (C) The HepG2 and Huh7 cells were exposed to vehicle or SP141 (0.5 μM) for 24 h, followed by immunofluorescence detection of MDM2. (D) The cells were treated with or without SP141 for 24 h, followed by exposure to a protein synthesis inhibitor, cycloheximide (CHX, 15 μg/mL). The protein levels of MDM2 and p53 were detected at the indicated times after exposure to CHX. (E) The HepG2 and Huh7 cells were treated with or without SP141 (0.5 μM) for 24 h. They were then exposed to MG-132 (25 μM), a proteasome inhibitor, for an additional 6 h. The protein levels of MDM2 and p53 were detected by Western blotting. (F) The cells were co-transfected with MDM2 and ubiquitin plasmids, followed by treatment with SP141 (0, 0.2 and 0.5 μM) for 24 h. Then the cell lysates were subjected to immunoprecipitation with an anti-MDM2 antibody. The ubiquitinated MDM2 was detected using an anti-ubiquitin antibody. (G) The cells were transfected with a wild-type MDM2 plasmid or a mutant MDM2 plasmid (C464A) without E3 ligase activity, followed by exposure to SP141 (0, 0.2 and 0.5 μM) for 24 h. The MDM2 protein levels were detected. All assays were performed in triplicate and repeated three times.
    Figure Legend Snippet: SP141 induces MDM2 degradation in human HCC cells. (A) HepG2 and Huh7 cells were exposed to 1 μM of SP141 for 2 h, followed by cellular thermal shift assays. (B) HCC cells were exposed to various concentrations of SP141 for 24 h. The protein expression levels of MDM2, p53, and p21 were determined. (C) The HepG2 and Huh7 cells were exposed to vehicle or SP141 (0.5 μM) for 24 h, followed by immunofluorescence detection of MDM2. (D) The cells were treated with or without SP141 for 24 h, followed by exposure to a protein synthesis inhibitor, cycloheximide (CHX, 15 μg/mL). The protein levels of MDM2 and p53 were detected at the indicated times after exposure to CHX. (E) The HepG2 and Huh7 cells were treated with or without SP141 (0.5 μM) for 24 h. They were then exposed to MG-132 (25 μM), a proteasome inhibitor, for an additional 6 h. The protein levels of MDM2 and p53 were detected by Western blotting. (F) The cells were co-transfected with MDM2 and ubiquitin plasmids, followed by treatment with SP141 (0, 0.2 and 0.5 μM) for 24 h. Then the cell lysates were subjected to immunoprecipitation with an anti-MDM2 antibody. The ubiquitinated MDM2 was detected using an anti-ubiquitin antibody. (G) The cells were transfected with a wild-type MDM2 plasmid or a mutant MDM2 plasmid (C464A) without E3 ligase activity, followed by exposure to SP141 (0, 0.2 and 0.5 μM) for 24 h. The MDM2 protein levels were detected. All assays were performed in triplicate and repeated three times.

    Techniques Used: Expressing, Immunofluorescence, Western Blot, Transfection, Immunoprecipitation, Plasmid Preparation, Mutagenesis, Activity Assay

    CRISPR/Cas9-mediated MDM2 knockout blocks SP141's activity in HCC cells. HepG2 and Huh7 cell lines with or without CRISPR/Cas9-mediated MDM2 knockout (sgMDM2) were exposed to various concentrations of SP141 for (A) 24 h for analyses of protein expression or (B) 72 h for cell viability measurement via MTT assays. (C) These cells were then treated with or without SP141 (0.05 μM) for 24 h, followed by measurement of the cell invasion via transwell cell invasion assays. All assays were performed in triplicate and repeated three times. (* P
    Figure Legend Snippet: CRISPR/Cas9-mediated MDM2 knockout blocks SP141's activity in HCC cells. HepG2 and Huh7 cell lines with or without CRISPR/Cas9-mediated MDM2 knockout (sgMDM2) were exposed to various concentrations of SP141 for (A) 24 h for analyses of protein expression or (B) 72 h for cell viability measurement via MTT assays. (C) These cells were then treated with or without SP141 (0.05 μM) for 24 h, followed by measurement of the cell invasion via transwell cell invasion assays. All assays were performed in triplicate and repeated three times. (* P

    Techniques Used: CRISPR, Knock-Out, Activity Assay, Expressing, MTT Assay

    39) Product Images from "Novel Broad-Spectrum Antiviral Inhibitors Targeting Host Factors Essential for Replication of Pathogenic RNA Viruses"

    Article Title: Novel Broad-Spectrum Antiviral Inhibitors Targeting Host Factors Essential for Replication of Pathogenic RNA Viruses

    Journal: Viruses

    doi: 10.3390/v12121423

    Validation of broad-spectrum activity and wide therapeutic window of the novel antiviral compounds. ( A ) Vero E6 cells were infected with SARS-CoV-2 (MOI 0.05), Huh7 cells were infected with CoV 229E (MOI 5), Vero cells were infected with EBOV (MOI 0.5), CCHFV (MOI 1) and SW13 cells were infected with HAZV (MOI 1). Infection was followed by treatment with 10 μM TH3289 for 24 h (SARS-CoV-2, CoV 229E, HAZV) or 48 h (EBOV, CCHFV) and end-point dilution assay was performed on Vero E6 (SARS-CoV-2), Vero cells (CCHFV, EBOV) or Huh7 cells (CoV 229E). Data are presented as mean ± SD from n = 2 (SARS-CoV-2, CoV 229E, CCHFV, HAZV) and n = 1 (EBOV) biological replicates. Statistical significance was determined using unpaired two-tailed t -tests. *** p
    Figure Legend Snippet: Validation of broad-spectrum activity and wide therapeutic window of the novel antiviral compounds. ( A ) Vero E6 cells were infected with SARS-CoV-2 (MOI 0.05), Huh7 cells were infected with CoV 229E (MOI 5), Vero cells were infected with EBOV (MOI 0.5), CCHFV (MOI 1) and SW13 cells were infected with HAZV (MOI 1). Infection was followed by treatment with 10 μM TH3289 for 24 h (SARS-CoV-2, CoV 229E, HAZV) or 48 h (EBOV, CCHFV) and end-point dilution assay was performed on Vero E6 (SARS-CoV-2), Vero cells (CCHFV, EBOV) or Huh7 cells (CoV 229E). Data are presented as mean ± SD from n = 2 (SARS-CoV-2, CoV 229E, CCHFV, HAZV) and n = 1 (EBOV) biological replicates. Statistical significance was determined using unpaired two-tailed t -tests. *** p

    Techniques Used: Activity Assay, Infection, End-point Dilution Assay, Two Tailed Test

    40) Product Images from "18β-Glycyrrhetinic acid protects against alpha-naphthylisothiocyanate-induced cholestasis through activation of the Sirt1/FXR signaling pathway"

    Article Title: 18β-Glycyrrhetinic acid protects against alpha-naphthylisothiocyanate-induced cholestasis through activation of the Sirt1/FXR signaling pathway

    Journal: Acta Pharmacologica Sinica

    doi: 10.1038/s41401-018-0110-y

    18b-GA activates FXR in vitro. After transfection with FXR plasmid DNA for 6 h, the dose-dependent response of Huh7 cells to 18b-GA exposure for 24 h with ( a ) and without ( b ) CDCA (50 μM), an effective FXR endogenous ligand, was measured. ( c ) Luciferase reporter assays confirmed that FXR is regulated by Sirt1. 18b-GA treatment significantly activated FXR, and FXR activity was significantly reduced by EX-527. SRT1720, a selective activator of human Sirt1, and EX-527, a potent and selective Sirt1 inhibitor, were used. Data are reported as the mean ± SEM ( n = 3). * P
    Figure Legend Snippet: 18b-GA activates FXR in vitro. After transfection with FXR plasmid DNA for 6 h, the dose-dependent response of Huh7 cells to 18b-GA exposure for 24 h with ( a ) and without ( b ) CDCA (50 μM), an effective FXR endogenous ligand, was measured. ( c ) Luciferase reporter assays confirmed that FXR is regulated by Sirt1. 18b-GA treatment significantly activated FXR, and FXR activity was significantly reduced by EX-527. SRT1720, a selective activator of human Sirt1, and EX-527, a potent and selective Sirt1 inhibitor, were used. Data are reported as the mean ± SEM ( n = 3). * P

    Techniques Used: In Vitro, Transfection, Plasmid Preparation, Luciferase, Activity Assay

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    CSMut1 design, modelling and infectivity. ( a ) The wild type E 250–270 and CSMut1 sequences are shown with mutated amino acids highlighted in red. ( b ) Wild type (on the left) and CSMut1 model (on the right) with E 250–270 in red. ( c ) Relative quantification of DV NS5 RNA in <t>Huh7.5</t> cells transfected with wild type and CSMut1 genomes at different time points. Cell associated RNA is shown in the left panel and supernatant extracted RNA in the right panel. ( d ) DV E-protein immunofluorescence staining of WT and CSMut1 120 hours after genome transfection in Huh7.5 cells (bars correspond to 32 μM).
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    Melatonin enhanced the sensitivity of <t>HCC</t> cells to chemotherapy by inducing lncRNA RAD51-AS1 expression. ( A – C ) The effects of melatonin combined with etoposide (VP16) on <t>Huh7</t> and HepG2 cell proliferation, migration and invasion with/without treatment with RAD51-AS1 siRNA (50 nM). p
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    86
    ATCC huh7 cells
    Filamin B knockdown inhibits the replication of HBV. ( A ) <t>Huh7</t> and HepG2 cells were transfected with siNC, filamin B-targeting siFLNB-1, siFLNB-2 or siFLNB-3 separately. Western blot analysis of filamin B was used to confirm the silencing efficiency. ( B ) Levels of HBV total RNAs and pgRNA in Huh7 cells were determined by real-time PCR. ( C ) Levels of HBV total RNAs and pgRNA in HepG2 cells were determined by real-time PCR. ( D ) Levels of HBeAg and HBsAg in culture medium supernatant of Huh7 cells were detected with ELISA. ( E ) Levels of HBeAg and HBsAg in culture medium supernatant of HepG2 cells were detected with ELISA. ( F ) HBV DNA was extracted at 96 h post transfection, and then subjected to Southern blotting. Western blot showed that filamin B was successfully knocked down. Co-transfection with pHBV1.3 and siNC was used as a loading control. Ratios were quantified by gray analysis with GeneTools from Syngene software (GeneGnome5). rcDNA, relaxed circular DNA; dsDNA, double stranded DNA; ssDNA, single stranded DNA. All results are shown as mean ± SD. (* P
    Huh7 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mcf 7  (ATCC)
    99
    ATCC mcf 7
    Cytotoxic effect of carboplatin, Fe 3 O 4 -NPs, and combination of carboplatin and Fe 3 O 4 -NPs on the Hep G2, <t>MCF-7,</t> and A549 cell lines. Cell viabilities are detected using MTT assay. Cells are incubated with various concentrations of the tested compound for 24 h at 37 °C. Results are illustrated as means ± standard deviations. Different letters represent significant differences ( p
    Mcf 7, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mcf 7/product/ATCC
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mcf 7 - by Bioz Stars, 2022-08
    99/100 stars
      Buy from Supplier

    Image Search Results


    CSMut1 design, modelling and infectivity. ( a ) The wild type E 250–270 and CSMut1 sequences are shown with mutated amino acids highlighted in red. ( b ) Wild type (on the left) and CSMut1 model (on the right) with E 250–270 in red. ( c ) Relative quantification of DV NS5 RNA in Huh7.5 cells transfected with wild type and CSMut1 genomes at different time points. Cell associated RNA is shown in the left panel and supernatant extracted RNA in the right panel. ( d ) DV E-protein immunofluorescence staining of WT and CSMut1 120 hours after genome transfection in Huh7.5 cells (bars correspond to 32 μM).

    Journal: Scientific Reports

    Article Title: Genome-wide analyses reveal a highly conserved Dengue virus envelope peptide which is critical for virus viability and antigenic in humans

    doi: 10.1038/srep36339

    Figure Lengend Snippet: CSMut1 design, modelling and infectivity. ( a ) The wild type E 250–270 and CSMut1 sequences are shown with mutated amino acids highlighted in red. ( b ) Wild type (on the left) and CSMut1 model (on the right) with E 250–270 in red. ( c ) Relative quantification of DV NS5 RNA in Huh7.5 cells transfected with wild type and CSMut1 genomes at different time points. Cell associated RNA is shown in the left panel and supernatant extracted RNA in the right panel. ( d ) DV E-protein immunofluorescence staining of WT and CSMut1 120 hours after genome transfection in Huh7.5 cells (bars correspond to 32 μM).

    Article Snippet: Briefly, Huh7.5 cells were transfected with wild type or CSMut1 RNA using Lipofectin, fixed after 120 hours, and stained with monoclonal anti-E antibody (4G2 - ATCC® HB-112™ ), using Alexa Fluor 488 rabbit anti-mouse IgG (H + L, Life Technologies) as a secondary antibody, and DAPI counter stain (Molecular Probes).

    Techniques: Infection, Transfection, Immunofluorescence, Staining

    Melatonin enhanced the sensitivity of HCC cells to chemotherapy by inducing lncRNA RAD51-AS1 expression. ( A – C ) The effects of melatonin combined with etoposide (VP16) on Huh7 and HepG2 cell proliferation, migration and invasion with/without treatment with RAD51-AS1 siRNA (50 nM). p

    Journal: Cancers

    Article Title: Melatonin Sensitizes Hepatocellular Carcinoma Cells to Chemotherapy Through Long Non-Coding RNA RAD51-AS1-Mediated Suppression of DNA Repair

    doi: 10.3390/cancers10090320

    Figure Lengend Snippet: Melatonin enhanced the sensitivity of HCC cells to chemotherapy by inducing lncRNA RAD51-AS1 expression. ( A – C ) The effects of melatonin combined with etoposide (VP16) on Huh7 and HepG2 cell proliferation, migration and invasion with/without treatment with RAD51-AS1 siRNA (50 nM). p

    Article Snippet: Cell Lines, Antibodies, Drug, siRNA and Plasmid Construction The HCC cell lines Huh7 and HepG2 were purchased from American Type Culture Collection (Manassas, VA, USA), which supplies authenticated cell lines.

    Techniques: Expressing, Migration

    Melatonin suppressed proliferation, migration and invasion capacities in HCC cells. ( A ) Huh7 and HepG2 cells were treated with 1 mM melatonin, and cell proliferation capacity was assessed at the indicated time points using an xCELLigence real-time cell analyzer. Mock: cells treated with DMEM medium. Vehicle: cells treated with DMSO. p

    Journal: Cancers

    Article Title: Melatonin Sensitizes Hepatocellular Carcinoma Cells to Chemotherapy Through Long Non-Coding RNA RAD51-AS1-Mediated Suppression of DNA Repair

    doi: 10.3390/cancers10090320

    Figure Lengend Snippet: Melatonin suppressed proliferation, migration and invasion capacities in HCC cells. ( A ) Huh7 and HepG2 cells were treated with 1 mM melatonin, and cell proliferation capacity was assessed at the indicated time points using an xCELLigence real-time cell analyzer. Mock: cells treated with DMEM medium. Vehicle: cells treated with DMSO. p

    Article Snippet: Cell Lines, Antibodies, Drug, siRNA and Plasmid Construction The HCC cell lines Huh7 and HepG2 were purchased from American Type Culture Collection (Manassas, VA, USA), which supplies authenticated cell lines.

    Techniques: Migration

    Melatonin suppressed the DNA repair capacity of HCC cells by inhibiting RAD51 expression. ( A ) A comet assay shows DNA repair activity after treatment with melatonin. Huh7 cells were treated with 200 µM etoposide (VP16) for 1 h, followed by treatment with different concentrations of melatonin in etoposide-free medium for 4 h. The cells were harvested and subjected to comet assays to detect DNA repair activity. The rows of panels present the results of three individual experiments. Quantification of cell repair activity is shown in ( B ). p

    Journal: Cancers

    Article Title: Melatonin Sensitizes Hepatocellular Carcinoma Cells to Chemotherapy Through Long Non-Coding RNA RAD51-AS1-Mediated Suppression of DNA Repair

    doi: 10.3390/cancers10090320

    Figure Lengend Snippet: Melatonin suppressed the DNA repair capacity of HCC cells by inhibiting RAD51 expression. ( A ) A comet assay shows DNA repair activity after treatment with melatonin. Huh7 cells were treated with 200 µM etoposide (VP16) for 1 h, followed by treatment with different concentrations of melatonin in etoposide-free medium for 4 h. The cells were harvested and subjected to comet assays to detect DNA repair activity. The rows of panels present the results of three individual experiments. Quantification of cell repair activity is shown in ( B ). p

    Article Snippet: Cell Lines, Antibodies, Drug, siRNA and Plasmid Construction The HCC cell lines Huh7 and HepG2 were purchased from American Type Culture Collection (Manassas, VA, USA), which supplies authenticated cell lines.

    Techniques: Expressing, Single Cell Gel Electrophoresis, Activity Assay

    Melatonin enhanced the sensitivity of HCC cells to chemotherapy and radiotherapy. ( A ) The proliferation capacity of Huh7 and HepG2 cells treated with 1 mM melatonin, 200 µM etoposide (VP16), or both was monitored using an xCELLigence real-time cell analyzer. p

    Journal: Cancers

    Article Title: Melatonin Sensitizes Hepatocellular Carcinoma Cells to Chemotherapy Through Long Non-Coding RNA RAD51-AS1-Mediated Suppression of DNA Repair

    doi: 10.3390/cancers10090320

    Figure Lengend Snippet: Melatonin enhanced the sensitivity of HCC cells to chemotherapy and radiotherapy. ( A ) The proliferation capacity of Huh7 and HepG2 cells treated with 1 mM melatonin, 200 µM etoposide (VP16), or both was monitored using an xCELLigence real-time cell analyzer. p

    Article Snippet: Cell Lines, Antibodies, Drug, siRNA and Plasmid Construction The HCC cell lines Huh7 and HepG2 were purchased from American Type Culture Collection (Manassas, VA, USA), which supplies authenticated cell lines.

    Techniques:

    Filamin B knockdown inhibits the replication of HBV. ( A ) Huh7 and HepG2 cells were transfected with siNC, filamin B-targeting siFLNB-1, siFLNB-2 or siFLNB-3 separately. Western blot analysis of filamin B was used to confirm the silencing efficiency. ( B ) Levels of HBV total RNAs and pgRNA in Huh7 cells were determined by real-time PCR. ( C ) Levels of HBV total RNAs and pgRNA in HepG2 cells were determined by real-time PCR. ( D ) Levels of HBeAg and HBsAg in culture medium supernatant of Huh7 cells were detected with ELISA. ( E ) Levels of HBeAg and HBsAg in culture medium supernatant of HepG2 cells were detected with ELISA. ( F ) HBV DNA was extracted at 96 h post transfection, and then subjected to Southern blotting. Western blot showed that filamin B was successfully knocked down. Co-transfection with pHBV1.3 and siNC was used as a loading control. Ratios were quantified by gray analysis with GeneTools from Syngene software (GeneGnome5). rcDNA, relaxed circular DNA; dsDNA, double stranded DNA; ssDNA, single stranded DNA. All results are shown as mean ± SD. (* P

    Journal: Virologica Sinica

    Article Title: Mechanisms and Effects on HBV Replication of the Interaction between HBV Core Protein and Cellular Filamin B

    doi: 10.1007/s12250-018-0023-4

    Figure Lengend Snippet: Filamin B knockdown inhibits the replication of HBV. ( A ) Huh7 and HepG2 cells were transfected with siNC, filamin B-targeting siFLNB-1, siFLNB-2 or siFLNB-3 separately. Western blot analysis of filamin B was used to confirm the silencing efficiency. ( B ) Levels of HBV total RNAs and pgRNA in Huh7 cells were determined by real-time PCR. ( C ) Levels of HBV total RNAs and pgRNA in HepG2 cells were determined by real-time PCR. ( D ) Levels of HBeAg and HBsAg in culture medium supernatant of Huh7 cells were detected with ELISA. ( E ) Levels of HBeAg and HBsAg in culture medium supernatant of HepG2 cells were detected with ELISA. ( F ) HBV DNA was extracted at 96 h post transfection, and then subjected to Southern blotting. Western blot showed that filamin B was successfully knocked down. Co-transfection with pHBV1.3 and siNC was used as a loading control. Ratios were quantified by gray analysis with GeneTools from Syngene software (GeneGnome5). rcDNA, relaxed circular DNA; dsDNA, double stranded DNA; ssDNA, single stranded DNA. All results are shown as mean ± SD. (* P

    Article Snippet: HEK 293T cells, Huh7 cells and HepG2 cells (from the American Type Culture Collection [ATCC]) were maintained in Dulbecco’s modified Eagle’s medium (Gibco, Shanghai, China) supplemented with 10% fetal bovine serum (Gibco) at 37 °C and 5% CO2 .

    Techniques: Transfection, Western Blot, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Southern Blot, Cotransfection, Software

    Interaction of filamin B and core protein promotes HBV replication. ( A ) Levels of HBV total RNAs and pgRNA in Huh7 cells were determined by real-time PCR. ( B ) HBV total RNAs and pgRNA levels in HepG2 cells were determined by real-time PCR. ( C ) Levels of HBeAg and HBsAg in culture medium supernatant of Huh7 cells were detected with ELISA. ( D ) Levels of HBeAg and HBsAg in culture medium supernatant of HepG2 cells were detected with ELISA. ( E ) HBV DNA was extracted at 96 h post-transfection and subjected to Southern blotting. Western blot showed that all proteins were overexpressed. Co-transfection with pHBV1.3, GFP and pXJ40-HA was used as a loading control. Ratios were quantified by gray analysis with GeneTools from Syngene software (GeneGnome5). HBV DNA marker bands are shown for relaxed circular DNA (rcDNA), double stranded DNA (dsDNA), and single stranded DNA (ssDNA). All results are shown as mean ± SD. (* P

    Journal: Virologica Sinica

    Article Title: Mechanisms and Effects on HBV Replication of the Interaction between HBV Core Protein and Cellular Filamin B

    doi: 10.1007/s12250-018-0023-4

    Figure Lengend Snippet: Interaction of filamin B and core protein promotes HBV replication. ( A ) Levels of HBV total RNAs and pgRNA in Huh7 cells were determined by real-time PCR. ( B ) HBV total RNAs and pgRNA levels in HepG2 cells were determined by real-time PCR. ( C ) Levels of HBeAg and HBsAg in culture medium supernatant of Huh7 cells were detected with ELISA. ( D ) Levels of HBeAg and HBsAg in culture medium supernatant of HepG2 cells were detected with ELISA. ( E ) HBV DNA was extracted at 96 h post-transfection and subjected to Southern blotting. Western blot showed that all proteins were overexpressed. Co-transfection with pHBV1.3, GFP and pXJ40-HA was used as a loading control. Ratios were quantified by gray analysis with GeneTools from Syngene software (GeneGnome5). HBV DNA marker bands are shown for relaxed circular DNA (rcDNA), double stranded DNA (dsDNA), and single stranded DNA (ssDNA). All results are shown as mean ± SD. (* P

    Article Snippet: HEK 293T cells, Huh7 cells and HepG2 cells (from the American Type Culture Collection [ATCC]) were maintained in Dulbecco’s modified Eagle’s medium (Gibco, Shanghai, China) supplemented with 10% fetal bovine serum (Gibco) at 37 °C and 5% CO2 .

    Techniques: Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Transfection, Southern Blot, Western Blot, Cotransfection, Software, Marker

    Filamin B interacts with core protein in different cell lines. Co-immunoprecipitation of filamin B and core protein is shown. Either FLNB-GFP plasmid or GFP vector (10 μg) was co-transfected with 10 μg Core-HA plasmid into HEK 293T, Huh7, and HepG2 cells. Filamin B was immunoprecipitated using GFP. Co-immunoprecipitated core protein was detected by immunoblotting with anti-HA antibodies (top). Western blot analysis of the precipitated filamin B and GFP using anti-GFP antibodies is shown (top). Cell lysates before immunoprecipitation were probed with anti-GFP and anti-HA antibodies (bottom).

    Journal: Virologica Sinica

    Article Title: Mechanisms and Effects on HBV Replication of the Interaction between HBV Core Protein and Cellular Filamin B

    doi: 10.1007/s12250-018-0023-4

    Figure Lengend Snippet: Filamin B interacts with core protein in different cell lines. Co-immunoprecipitation of filamin B and core protein is shown. Either FLNB-GFP plasmid or GFP vector (10 μg) was co-transfected with 10 μg Core-HA plasmid into HEK 293T, Huh7, and HepG2 cells. Filamin B was immunoprecipitated using GFP. Co-immunoprecipitated core protein was detected by immunoblotting with anti-HA antibodies (top). Western blot analysis of the precipitated filamin B and GFP using anti-GFP antibodies is shown (top). Cell lysates before immunoprecipitation were probed with anti-GFP and anti-HA antibodies (bottom).

    Article Snippet: HEK 293T cells, Huh7 cells and HepG2 cells (from the American Type Culture Collection [ATCC]) were maintained in Dulbecco’s modified Eagle’s medium (Gibco, Shanghai, China) supplemented with 10% fetal bovine serum (Gibco) at 37 °C and 5% CO2 .

    Techniques: Immunoprecipitation, Plasmid Preparation, Transfection, Western Blot

    Cytotoxic effect of carboplatin, Fe 3 O 4 -NPs, and combination of carboplatin and Fe 3 O 4 -NPs on the Hep G2, MCF-7, and A549 cell lines. Cell viabilities are detected using MTT assay. Cells are incubated with various concentrations of the tested compound for 24 h at 37 °C. Results are illustrated as means ± standard deviations. Different letters represent significant differences ( p

    Journal: Journal of Fungi

    Article Title: Thiolation of Myco-Synthesized Fe3O4-NPs: A Novel Promising Tool for Penicillium expansium Laccase Immobilization to Decolorize Textile Dyes and as an Application for Anticancer Agent

    doi: 10.3390/jof8010071

    Figure Lengend Snippet: Cytotoxic effect of carboplatin, Fe 3 O 4 -NPs, and combination of carboplatin and Fe 3 O 4 -NPs on the Hep G2, MCF-7, and A549 cell lines. Cell viabilities are detected using MTT assay. Cells are incubated with various concentrations of the tested compound for 24 h at 37 °C. Results are illustrated as means ± standard deviations. Different letters represent significant differences ( p

    Article Snippet: MCF-7 (human breast cancer cells), HepG2 (human hepatocellular carcinoma cells), and A549 cell lines were brought from ATCC via the holding company for biological products and vaccines (VACSERA), Cairo, Egypt.

    Techniques: MTT Assay, Incubation