anti-actin Search Results


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  • 99
    Millipore monoclonal anti beta actin antibody
    Parkin truncating variants have reduced protein expression. ( A ) Schematic representation of parkin (NP_004553) functional domains and location of the frameshift variants identified in Portuguese patients. ( B ) Analysis of protein expression of EGFP-tagged parkin clones in HEK293T cells by immunoblotting with anti-EGFP antibody. <t>Beta-actin</t> was used as loading control. Original blots are presented in Supplementary Fig. S2 . Quantification data (graph) are presented as the mean ± SD of three independent experiments; **p
    Monoclonal Anti Beta Actin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 35169 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti β actin
    MUC1 is widely overexpressed in NSCLC cells, correlating with STAT3 activation. (A) Protein expression of MUC1-C and total and Tyr705 phosphorylated STAT3 levels were determined in 14 human NSCLC cell lines by Western blot analyses. Equal loading and transfer were shown by repeat probing with <t>β-actin.</t> (B) mRNA levels of MUC1 in a subset of cells were evaluated with real-time RT-PCR. MUC1 mRNA expression levels in each cell line were normalized to GAPDH mRNA, with expression in A549 cells set to an arbitrary value of 1. * P
    Anti β Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 26921 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Cell Signaling Technology Inc β actin
    Loss of Ei24 impairs glucose homeostasis. A , expression of Ei24 in islets from ob/ob mice, GK rats at 4 months of age, and C57BL/6 mice fed an HFD for 2 weeks and 8 weeks. <t>β-Actin</t> served as the loading control. CD , chow diet. B , total RNA was prepared from islets of WT and KO mice at 8 weeks of age. The transcription levels of Ei24 mRNA are normalized to β-actin mRNA. The results are representative of three individual experiments. C , Western blotting of Ei24 protein from isolated islets (300 islets/group) from WT and KO mice at 12 weeks of age. β-Actin served as the loading control. D , weight curves for WT and KO mice. The mean ± S.D. ( error bars ) of 15 mice is shown. E , concentration of the basic glucose curve for WT and KO mice. The mean ± S.D. of 10 mice is shown. F and G , glucose tolerance test ( GTT ) results of 10-week-old male ( F ) and female mice ( G ). Solid lines , WT mice ( n = 8); dashed lines , KO mice ( n = 8). H and I , ITT results of 10-week-old male ( H ) and female mice ( I ). Solid lines , WT mice ( n = 8); dashed lines , KO mice ( n = 8). J , in vivo GSIS detection in WT and KO mice. The results are representative of five replicates for each group. K , in vitro GSIS from isolated islets (70/group) of WT and KO mice by a fast digital perfusion system. The results are representative of three individual experiments. Data are expressed as the mean ± S.D. *, p
    β Actin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 38407 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Santa Cruz Biotechnology β actin
    Knockdown of AQP5 suppressed the ability of migration and invasion in HCT116 and SW480 cells. a The ability of migration was measured by scratch wound healing assay after knockdown of AQP5, the migration rate was determined. b The invasiveness of HCT116 and SW480 cells with and without AQP5 silencing was evaluated by transwell assay, invasive cells were counted and expressed as fold change compared to the Mock group. c The expression levels of MMP-2 and MMP-9 were detected by immunoblotting after knockdown of AQP5, the bands were semi-quantified by densitometry, normalized to <t>β-actin</t> expression and expressed as fold change compared with the Mock group. d The activities of MMP-2 and MMP-9 in AQP5-silenced and control cells were assessed by gelatin zymography. Data were presented as the mean ± SD. * p
    β Actin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 97/100, based on 50954 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti beta actin
    Nuclear import of the fusion protein GST-GFP-NLS in the permeabilized cells. Cells permeabilized with 30 µg/mL digitonin (Dig30) and non-permeabilized cells (Control) were incubated with GST-GFP-NLS in the absence (−Extract) or presence (+Extract) of Xenopus egg extract supplemented with ATP for 1 h at 25 °C. The incorporation of GST-GFP-NLS was assessed by green fluorescence. Cell nuclei were counterstained with Hoechst 33243. Note that without egg extract, the nuclei are not labelled (arrows). Due to light scattering of the fluorescence in the cytoplasm, the nuclei appear smaller than they are. The egg extract restored nuclear import in the permeabilized cells. These pictures are representative of five independent replicates. Scale bar = 20 µm. Detection of importin alpha-1 (55 kDa) ( B ) and Karyopherin <t>beta</t> -1 (KPNB1–97 kDa) ( C ) in Xenopus egg extract by western blot in the presence (+) or in absence (−) of the anti- Xenopus importin alpha-1 antibody (clone 15) and the anti-rat KPNB1 antibody (KPNB1-clone 23). Beta actin was used as a loading control. M: size markers. The cropped blots came from the same gels and were analyzed with the same exposure times (B:1 sec; C:1 min).
    Anti Beta Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4852 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam β actin
    Reduced PAX8 expression leads to decreased expression of BCL2 and WT1. (A) The PAX8 -knockdown (siPAX8) in the A172 glioma cell line by siRNA (PAX8-1) produced a reduction in the BCL2 expression levels. Cells lysates were prepared 36 hours after siRNA transfection, and the PAX8, BCL2, p53, and <t>β-actin</t> (loading control) expression levels were measured by western blot. For controls, A172 cells were transfected with mock-treated (Moc), non-targeting siRNAs (NT1, NT2, and NT3) and scrambled s8-1 siRNA (scPAX8). To ensure the reduction in the glioma cell growth rate associated with the PAX8 -knockdown was not due to p53 function, p53 was also knocked down in A172 cells (sip53) independently or in combination with a PAX8 siRNA (siPAX8 p53). (B ) The PAX8 -knockdown (siPAX8) in the A172 glioma cell line by siRNA (PAX8-1) produced a reduction in the WT1 expression levels. (C) The BCL2 -knockdown produced a similar reduction in the cell growth rate compared to PAX8 -knockdown in the A172 glioma cell line. Cells were transfected with a BCL2 siRNA (siBCL2) or a PAX8 siRNA (PAX8-1, siPAX8). For controls, A172 cells were mock-transfected (Moc) or transfected with non-targeting siRNAs (NT1 and NT3). The percentage of live cells was determined by the trypan blue exclusion assay every 24 hours post-transfection. ( Insert ) Western blotting shows the BCL2-knockdown with a BCL2 siRNA and no BCL2-knockdown in controls; the loading control is β-actin.
    β Actin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 25356 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti β actin
    Zey suppresses the Wnt/β-catenin pathway in DU145 cells. (A) DU145 cells were administrated with Zey at 0 (control), 10 (Zey1), 20 (Zey2) and 40 (Zey3) µmol/l for 48 h. Western blot analysis was performed to measure the protein expression of wnt5a, β-catenin and cyclin D1. (B) Quantification of protein was executed using GraphPad Prism software. <t>β-actin</t> was regarded as the internal control. Gray value was assessed and calculated using Quantity One software. ^ P
    Anti β Actin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 8076 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore monoclonal anti actin alpha smooth muscle
    Immunocytochemical characterization. Confluent HUVEC cell layers after 24 h of NAC supplemented culture. In 15 mM NAC enriched cultivation, endothelial cells changed their morphology from typical cobblestone pattern (a–d) to an elongated, quasi-fusiform configuration (i–l). A slight cellular rearrangement was already detectable at 10 mM (e–h). Cells were positive for common endothelial cell markers CD31 (PECAM-1) and Von-Willebrand-Factor (vWF). Nuclei were counterstained with DAPI. All cells were negative for <t>alpha</t> smooth muscle actin (αSMA), a common myofibroblast marker (scale bar: 100 µ m).
    Monoclonal Anti Actin Alpha Smooth Muscle, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4985 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Abcam anti beta actin antibody
    CD47 is highly expressed in osteosarcoma Quantitative RT-PCR A. and immunoblot analysis B. showed CD47 expression in ten freshly isolated osteosarcoma tissues and their adjacent nontumorous tissues. <t>Beta-actin</t> was treated as the reference control. CD47 immunostaining C. showed higher of CD47 expression in osteosarcoma of the same samples with or without metastasis and their adjacent nontumorous tissues. The tissues were counterstained with DAPI Fluorescent Stain. Representative images are shown here. (magnification × 100). D. Flow-cytometry analysis of CD47 and CD44 expressions on osteosarcoma tumor cells and quantification of CD44 cells also expressing CD47.
    Anti Beta Actin Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 98/100, based on 1248 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Parkin truncating variants have reduced protein expression. ( A ) Schematic representation of parkin (NP_004553) functional domains and location of the frameshift variants identified in Portuguese patients. ( B ) Analysis of protein expression of EGFP-tagged parkin clones in HEK293T cells by immunoblotting with anti-EGFP antibody. Beta-actin was used as loading control. Original blots are presented in Supplementary Fig. S2 . Quantification data (graph) are presented as the mean ± SD of three independent experiments; **p

    Journal: Scientific Reports

    Article Title: Parkin truncating variants result in a loss-of-function phenotype

    doi: 10.1038/s41598-019-52534-6

    Figure Lengend Snippet: Parkin truncating variants have reduced protein expression. ( A ) Schematic representation of parkin (NP_004553) functional domains and location of the frameshift variants identified in Portuguese patients. ( B ) Analysis of protein expression of EGFP-tagged parkin clones in HEK293T cells by immunoblotting with anti-EGFP antibody. Beta-actin was used as loading control. Original blots are presented in Supplementary Fig. S2 . Quantification data (graph) are presented as the mean ± SD of three independent experiments; **p

    Article Snippet: Antibodies Primary antibodies: mouse monoclonal anti-EGFP antibody (Abnova, MAB1765), mouse monoclonal anti-GFP antibody (Rockland, 600-301-215), mouse monoclonal anti-beta-actin (Sigma-Aldrich, A5441), mouse monoclonal anti-GM130 (BD Biosciences, 610822), mouse monoclonal anti-caspase 3 (Cell Signaling, 9668), mouse monoclonal anti-HDAC2 (Santa Cruz Biotechnology, sc-9959), rabbit polyclonal anti-histone H3 (Abcam, ab1791), mouse monoclonal anti-p62 (Proteintech, 66184-1-Ig), mouse monoclonal anti-TOM20 (BD Biosciences, 612278).

    Techniques: Expressing, Functional Assay, Clone Assay

    Fyn regulates IP 3 -mediated calcium responses. (A) WEHI 7.2 T cells were transfected with Fyn siRNAs (or non-targeting control siRNAs) as described in materials and methods. Fyn levels were measured by western blotting 24 hours post-transfection. β-actin

    Journal: Autophagy

    Article Title: Glucocorticoids downregulate Fyn and inhibit IP3-mediated calcium signaling to promote autophagy in T lymphocytes

    doi: 10.4161/auto.6.7.13290

    Figure Lengend Snippet: Fyn regulates IP 3 -mediated calcium responses. (A) WEHI 7.2 T cells were transfected with Fyn siRNAs (or non-targeting control siRNAs) as described in materials and methods. Fyn levels were measured by western blotting 24 hours post-transfection. β-actin

    Article Snippet: The following antibodies were used in this study: Fyn (Santa Cruz Biotechnology, sc-16), Lck (Santa Cruz Biotechnology, sc-433), anti-mouse CD3ε (BD Biosciences, 145-2C11), IP3 R3 (BD Biosciences, 610312), β-actin (Sigma-Aldrich, A-5441), p62 (Novus Biologicals, 8878-M03), LC3 (Novus Biologicals, NB100-2220), IP3 R1 (Novus Biologicals, NB120-5908), IP3 R2 (Novus Biologicals, NB100-2466), phospho-S6 kinase (Thr389) (Cell Signaling Technology, 9205), phospho-4EBP1 (Ser65) (Cell Signaling Technology, 9451), Total S6 kinase (Cell Signaling Technology, 9202), Total 4EBP1 (Cell Signaling Technology, 9452).

    Techniques: Transfection, Western Blot

    MUC1 is widely overexpressed in NSCLC cells, correlating with STAT3 activation. (A) Protein expression of MUC1-C and total and Tyr705 phosphorylated STAT3 levels were determined in 14 human NSCLC cell lines by Western blot analyses. Equal loading and transfer were shown by repeat probing with β-actin. (B) mRNA levels of MUC1 in a subset of cells were evaluated with real-time RT-PCR. MUC1 mRNA expression levels in each cell line were normalized to GAPDH mRNA, with expression in A549 cells set to an arbitrary value of 1. * P

    Journal: International journal of oncology

    Article Title: MUC1 is a downstream target of STAT3 and regulates lung cancer cell survival and invasion

    doi:

    Figure Lengend Snippet: MUC1 is widely overexpressed in NSCLC cells, correlating with STAT3 activation. (A) Protein expression of MUC1-C and total and Tyr705 phosphorylated STAT3 levels were determined in 14 human NSCLC cell lines by Western blot analyses. Equal loading and transfer were shown by repeat probing with β-actin. (B) mRNA levels of MUC1 in a subset of cells were evaluated with real-time RT-PCR. MUC1 mRNA expression levels in each cell line were normalized to GAPDH mRNA, with expression in A549 cells set to an arbitrary value of 1. * P

    Article Snippet: Anti-β-actin and donkey anti-rabbit IgG HRP-conjugated secondary antibody were purchased from Sigma (St. Louis, MO) and Amersham Biosciences (Piscataway, NJ), respectively.

    Techniques: Activation Assay, Expressing, Western Blot, Quantitative RT-PCR

    Effects of MUC1 knockdown or overexpression on multiple cell signals in NSCLC cells. (A) H358, HCC827, and H441 cells were exposed to siRNA for 72 h, and (B) A549 cells were transfected with MUC1-C plasmid for 48 h followed by measurement of phosphorylated tyrosine STAT3, Akt, Src, FAK, and apoptotic-related proteins by Western blot analysis. Equal loading and transfer were shown by repeat probing with β-actin.

    Journal: International journal of oncology

    Article Title: MUC1 is a downstream target of STAT3 and regulates lung cancer cell survival and invasion

    doi:

    Figure Lengend Snippet: Effects of MUC1 knockdown or overexpression on multiple cell signals in NSCLC cells. (A) H358, HCC827, and H441 cells were exposed to siRNA for 72 h, and (B) A549 cells were transfected with MUC1-C plasmid for 48 h followed by measurement of phosphorylated tyrosine STAT3, Akt, Src, FAK, and apoptotic-related proteins by Western blot analysis. Equal loading and transfer were shown by repeat probing with β-actin.

    Article Snippet: Anti-β-actin and donkey anti-rabbit IgG HRP-conjugated secondary antibody were purchased from Sigma (St. Louis, MO) and Amersham Biosciences (Piscataway, NJ), respectively.

    Techniques: Over Expression, Transfection, Plasmid Preparation, Western Blot

    Effect of TSN on expression of HCV replicon. (A) HCV replicon cells were treated with various concentrations of TSN for 48 h. Replication levels of HCV RNA were analyzed by luciferase assay. Bars indicate luciferase activities relative to that of the drug-negative control. (B) Cell viability was determined by MTS assay. Bars indicate the value relative to that of the drug-negative control. (C) Western blotting analyses. The expression of NS5A and beta-actin was detected using anti-NS5A and anti-beta-actin antibodies. Densitometry of NS5A protein was performed, and the result is indicated as a percentage of the result for the drug-negative control. The assay was repeated three times, and a representative result is shown. (D) A bicistronic reporter gene plasmid, pCIneo-Rluc-IRES-Fluc, was transfected into Huh7 cells. The cells were cultured with TSN at the concentrations indicated, and dual luciferase activities were measured after 24 h of treatment. Values are displayed as ratios of Fluc to Rluc. In panels A, B, and D, the assays were done in triplicate and repeated three times. Error bars indicate means ± SDs.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Inhibitory Effect of a Triterpenoid Compound, with or without Alpha Interferon, on Hepatitis C Virus Infection ▿Inhibitory Effect of a Triterpenoid Compound, with or without Alpha Interferon, on Hepatitis C Virus Infection ▿ †

    doi: 10.1128/AAC.01780-10

    Figure Lengend Snippet: Effect of TSN on expression of HCV replicon. (A) HCV replicon cells were treated with various concentrations of TSN for 48 h. Replication levels of HCV RNA were analyzed by luciferase assay. Bars indicate luciferase activities relative to that of the drug-negative control. (B) Cell viability was determined by MTS assay. Bars indicate the value relative to that of the drug-negative control. (C) Western blotting analyses. The expression of NS5A and beta-actin was detected using anti-NS5A and anti-beta-actin antibodies. Densitometry of NS5A protein was performed, and the result is indicated as a percentage of the result for the drug-negative control. The assay was repeated three times, and a representative result is shown. (D) A bicistronic reporter gene plasmid, pCIneo-Rluc-IRES-Fluc, was transfected into Huh7 cells. The cells were cultured with TSN at the concentrations indicated, and dual luciferase activities were measured after 24 h of treatment. Values are displayed as ratios of Fluc to Rluc. In panels A, B, and D, the assays were done in triplicate and repeated three times. Error bars indicate means ± SDs.

    Article Snippet: The antibodies used were mouse anti-NS5A (BioDesign, ME), rabbit anti-signal transducer and activator of transcription 1 (anti-STAT1) p84/p91, rabbit anti-phospho-STAT1 (Tyr 701), rabbit anti-STAT2, rabbit anti-phospho-STAT2 (Tyr 690) (Santa Cruz, CA), and anti-beta-actin antibody (Sigma).

    Techniques: Expressing, Luciferase, Negative Control, MTS Assay, Western Blot, Plasmid Preparation, Transfection, Cell Culture

    ISRE reporter screening and aberrant pathway of α-IFN. (A) Pretreatment with TSN. Huh7 cells transfected with a reporter gene (pISRE-Luc and pRL-CMV) were pretreated with TSN (0 or 100 nM) for 0, 24, or 48 h, followed by treatment with α-IFN (0 or 100 IU/ml). Six hours later, the relative ISRE-luciferase activity ( n = 4) was determined as described in Materials and Methods. The data are expressed as means ± SDs and are a representative example of the data from three similar experiments. (B) Pretreatment with TSN at the concentrations indicated for 24 h, followed by treatment with α-IFN (0 to 100 IU/ml). The ISRE reporter assay was performed as described for panel A. (C) Type I IFN-induced antiviral ISG expression in Huh7 cells. Huh7 cells were treated with TSN for 24 h, followed by treatment with α-IFN at 100 IU/ml for 24 h. The total cellular RNA was then isolated for real-time RT-PCR analysis of the mRNAs of 25AS, MxA, p56, viperin, and ISG20. Beta-actin was used as a control. The data are expressed as means ± SDs and are a representative example of data from three similar experiments. *, P

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Inhibitory Effect of a Triterpenoid Compound, with or without Alpha Interferon, on Hepatitis C Virus Infection ▿Inhibitory Effect of a Triterpenoid Compound, with or without Alpha Interferon, on Hepatitis C Virus Infection ▿ †

    doi: 10.1128/AAC.01780-10

    Figure Lengend Snippet: ISRE reporter screening and aberrant pathway of α-IFN. (A) Pretreatment with TSN. Huh7 cells transfected with a reporter gene (pISRE-Luc and pRL-CMV) were pretreated with TSN (0 or 100 nM) for 0, 24, or 48 h, followed by treatment with α-IFN (0 or 100 IU/ml). Six hours later, the relative ISRE-luciferase activity ( n = 4) was determined as described in Materials and Methods. The data are expressed as means ± SDs and are a representative example of the data from three similar experiments. (B) Pretreatment with TSN at the concentrations indicated for 24 h, followed by treatment with α-IFN (0 to 100 IU/ml). The ISRE reporter assay was performed as described for panel A. (C) Type I IFN-induced antiviral ISG expression in Huh7 cells. Huh7 cells were treated with TSN for 24 h, followed by treatment with α-IFN at 100 IU/ml for 24 h. The total cellular RNA was then isolated for real-time RT-PCR analysis of the mRNAs of 25AS, MxA, p56, viperin, and ISG20. Beta-actin was used as a control. The data are expressed as means ± SDs and are a representative example of data from three similar experiments. *, P

    Article Snippet: The antibodies used were mouse anti-NS5A (BioDesign, ME), rabbit anti-signal transducer and activator of transcription 1 (anti-STAT1) p84/p91, rabbit anti-phospho-STAT1 (Tyr 701), rabbit anti-STAT2, rabbit anti-phospho-STAT2 (Tyr 690) (Santa Cruz, CA), and anti-beta-actin antibody (Sigma).

    Techniques: Transfection, Luciferase, Activity Assay, Reporter Assay, Expressing, Isolation, Quantitative RT-PCR

    Suppression of HCV RNA replication by TSN combined with α-IFN. (A and B) Luciferase activity (A, absolute value; B, relative value). Huh7/Rep-Feo cells, which constitutively express an HCV replicon, enable the quantification of replication levels through the measurement of luciferase activity. Absolute and relative dose-response curves in the presence of 24 h of pretreatment of various concentrations of TSN (0, 0.01, 0.03 μg/ml) and α-IFN (0, 100 IU/ml). (A) Bars indicate luciferase activities. (B) Bars indicate luciferase activities relative to the activity of each α-IFN-negative control. Luciferase assays were performed in triplicate. Error bars indicate means ± SDs. (C) MTS assay of Huh7/Rep-Feo cells cultured with the indicated concentrations of TSN and α-IFN. The assays were done in triplicate and repeated three times. Error bars indicate means ± SDs. (D) Western blotting. Ten micrograms of total cellular protein was separated by polyacrylamide gel electrophoresis and transferred onto the membrane. Monoclonal anti-NS5A antibody or an anti-beta-actin antibody was used as the primary antibody. Densitometry of NS5A or beta-actin protein was performed and the result is indicated as a percentage of that for the drug-negative control. The assay was repeated three times, and representative results are shown. (E) Dose-inhibition curves of α-IFN and TSN when they were combined at the indicated ratios, adjusted by the EC 50 of the individual drug. Assays were done in triplicate, and mean values were plotted and indicated as means ± SDs. (F) Graphical representation of the isobologram analysis. For each drug combination in panel E, the EC 50 s of α-IFN and TSN for inhibition of HCV replication were plotted against the fractional concentrations of α-IFN and TSN, which are indicated on the x and y axes, respectively. A theoretical line of additivity is drawn between the EC 50 for each drug alone. All of the fractional EC 50 plots for the TSN and α-IFN combinations fell below the line of additivity, indicating synergy.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Inhibitory Effect of a Triterpenoid Compound, with or without Alpha Interferon, on Hepatitis C Virus Infection ▿Inhibitory Effect of a Triterpenoid Compound, with or without Alpha Interferon, on Hepatitis C Virus Infection ▿ †

    doi: 10.1128/AAC.01780-10

    Figure Lengend Snippet: Suppression of HCV RNA replication by TSN combined with α-IFN. (A and B) Luciferase activity (A, absolute value; B, relative value). Huh7/Rep-Feo cells, which constitutively express an HCV replicon, enable the quantification of replication levels through the measurement of luciferase activity. Absolute and relative dose-response curves in the presence of 24 h of pretreatment of various concentrations of TSN (0, 0.01, 0.03 μg/ml) and α-IFN (0, 100 IU/ml). (A) Bars indicate luciferase activities. (B) Bars indicate luciferase activities relative to the activity of each α-IFN-negative control. Luciferase assays were performed in triplicate. Error bars indicate means ± SDs. (C) MTS assay of Huh7/Rep-Feo cells cultured with the indicated concentrations of TSN and α-IFN. The assays were done in triplicate and repeated three times. Error bars indicate means ± SDs. (D) Western blotting. Ten micrograms of total cellular protein was separated by polyacrylamide gel electrophoresis and transferred onto the membrane. Monoclonal anti-NS5A antibody or an anti-beta-actin antibody was used as the primary antibody. Densitometry of NS5A or beta-actin protein was performed and the result is indicated as a percentage of that for the drug-negative control. The assay was repeated three times, and representative results are shown. (E) Dose-inhibition curves of α-IFN and TSN when they were combined at the indicated ratios, adjusted by the EC 50 of the individual drug. Assays were done in triplicate, and mean values were plotted and indicated as means ± SDs. (F) Graphical representation of the isobologram analysis. For each drug combination in panel E, the EC 50 s of α-IFN and TSN for inhibition of HCV replication were plotted against the fractional concentrations of α-IFN and TSN, which are indicated on the x and y axes, respectively. A theoretical line of additivity is drawn between the EC 50 for each drug alone. All of the fractional EC 50 plots for the TSN and α-IFN combinations fell below the line of additivity, indicating synergy.

    Article Snippet: The antibodies used were mouse anti-NS5A (BioDesign, ME), rabbit anti-signal transducer and activator of transcription 1 (anti-STAT1) p84/p91, rabbit anti-phospho-STAT1 (Tyr 701), rabbit anti-STAT2, rabbit anti-phospho-STAT2 (Tyr 690) (Santa Cruz, CA), and anti-beta-actin antibody (Sigma).

    Techniques: Luciferase, Activity Assay, Negative Control, MTS Assay, Cell Culture, Western Blot, Polyacrylamide Gel Electrophoresis, Inhibition

    Antibody targeting of sAPP-α promotes amyloidogenic APP processing in vivo . PSAPP mice (3 female mice per group) at 8 months of age were intracerebroventricular (i.c.v.) injected with 2B3 antibody or isotype-matched IgG 2b control antibody at 5 μg/mL and sacrificed at 48 h after the injection. ( a ) Mouse brain homogenates were prepared from these mice and subjected to IB analysis for APP processing. IB using 6E10 antibody shows total APP and three bands corresponding to β-CTF and soluble Aβ species 38 and 40. Densitometry analysis shows the ratios of Aβ 40 to β-actin ( b ) and β-CTF to β-actin ( c ). A t test revealed significant differences in either ratio of Aβ 40 to β-actin or β-CTF to β-actin between 2B3 antibody- and control IgG 2b antibody-i.c.v. injected PSAPP mice. (*** P

    Journal: Nature communications

    Article Title: sAPP-? modulates ?-secretase activity and amyloid-? generation

    doi: 10.1038/ncomms1781

    Figure Lengend Snippet: Antibody targeting of sAPP-α promotes amyloidogenic APP processing in vivo . PSAPP mice (3 female mice per group) at 8 months of age were intracerebroventricular (i.c.v.) injected with 2B3 antibody or isotype-matched IgG 2b control antibody at 5 μg/mL and sacrificed at 48 h after the injection. ( a ) Mouse brain homogenates were prepared from these mice and subjected to IB analysis for APP processing. IB using 6E10 antibody shows total APP and three bands corresponding to β-CTF and soluble Aβ species 38 and 40. Densitometry analysis shows the ratios of Aβ 40 to β-actin ( b ) and β-CTF to β-actin ( c ). A t test revealed significant differences in either ratio of Aβ 40 to β-actin or β-CTF to β-actin between 2B3 antibody- and control IgG 2b antibody-i.c.v. injected PSAPP mice. (*** P

    Article Snippet: Other antibodies include: mouse monoclonal BACE1 antibody (1 mg/mL; Millipore, Billerica, MA); rabbit polyclonal BACE1 antibody and APP C-terminal antibody (500 μg/mL; EMD Biosciences, La Jolla, CA); APP N-terminus antibody (100 μg/mL, Millopore); Aβ17-24 antibody (1 mg/mL; 4G8, Covance) and Aβ1-12 antibody (BAM10, 500μg/mL; Sigma-Aldrich, St. Louis, MO); β-actin antibody (100 μg/mL; Sigma-Aldrich); rabbit anti-APP C-terminus polyclonal antibody (500 μg/mL; pAb369) generously provided by Dr. Sam Gandy; rabbit anti-APP C-terminus polyclonal antibody (pAb751/770, 100 μg/mL; Calbiochem); rabbit polyclonal oligomer/conformational (OC) antibody (500 μg/mL) was provided by Dr. Suhail Rasool and Dr. Charles G. Glabe at University of California.

    Techniques: In Vivo, Mouse Assay, Injection

    Immunodepleted sAPP-α attenuates sAPP-α effects on APP modulation in vitro. Active hsAPP-α protein at 1 nM was incubated with an anti-C-terminal sAPP-α specific 2B3 antibody or isotype-matched IgG 2b control antibody at 2.5 and 5 μ/mL at 37°C for 30 min. The protein-G sepharose beads were used to pull-down sAPP-α antibody and isotype-matched IgG 2b control, as well as, their associated proteins. This was followed by high-speed centrifugation at 15,000 g for 10 min, and the supernatants were collected and determined to be IgG antibody free by IB analysis using an antibody against IgG 2b . These supernatants were used to treat CHO cells expressing human wild-type APP (CHO/APP wt ) for 12 h. Cell cultured supernatants were collected and subjected to ( a ) Aβ IB analysis using 6E10 antibody and ( b ) the Aβ ELISA results are represented as the mean ± SD of picograms of Aβ 40 or Aβ 42 per milligram of total intracellular protein after the 2B3- or control IgG 2b -immunodepleted hsAPP-α protein treatment. ( c ) Cell lysates were prepared and APP CTFs were analyzed by IB using pAb751/770 (C-APP). ( d ) Relative ratio (mean ± SD) of β-CTF to β-actin was calculated by densitometry analysis. For b and d , the results as presented are representative of two independent experiments with n = 3 for each condition. One-way ANOVA followed by post hoc comparison revealed significant differences between 2B3-immunodepleted hsAPP-α protein treatment at either 2.5 or 5 μg/mL and control IgG 2b -immunodepleted hsAPP-α protein as measured by either Aβ levels or ratio of β-CTF to β-actin. (*** P

    Journal: Nature communications

    Article Title: sAPP-? modulates ?-secretase activity and amyloid-? generation

    doi: 10.1038/ncomms1781

    Figure Lengend Snippet: Immunodepleted sAPP-α attenuates sAPP-α effects on APP modulation in vitro. Active hsAPP-α protein at 1 nM was incubated with an anti-C-terminal sAPP-α specific 2B3 antibody or isotype-matched IgG 2b control antibody at 2.5 and 5 μ/mL at 37°C for 30 min. The protein-G sepharose beads were used to pull-down sAPP-α antibody and isotype-matched IgG 2b control, as well as, their associated proteins. This was followed by high-speed centrifugation at 15,000 g for 10 min, and the supernatants were collected and determined to be IgG antibody free by IB analysis using an antibody against IgG 2b . These supernatants were used to treat CHO cells expressing human wild-type APP (CHO/APP wt ) for 12 h. Cell cultured supernatants were collected and subjected to ( a ) Aβ IB analysis using 6E10 antibody and ( b ) the Aβ ELISA results are represented as the mean ± SD of picograms of Aβ 40 or Aβ 42 per milligram of total intracellular protein after the 2B3- or control IgG 2b -immunodepleted hsAPP-α protein treatment. ( c ) Cell lysates were prepared and APP CTFs were analyzed by IB using pAb751/770 (C-APP). ( d ) Relative ratio (mean ± SD) of β-CTF to β-actin was calculated by densitometry analysis. For b and d , the results as presented are representative of two independent experiments with n = 3 for each condition. One-way ANOVA followed by post hoc comparison revealed significant differences between 2B3-immunodepleted hsAPP-α protein treatment at either 2.5 or 5 μg/mL and control IgG 2b -immunodepleted hsAPP-α protein as measured by either Aβ levels or ratio of β-CTF to β-actin. (*** P

    Article Snippet: Other antibodies include: mouse monoclonal BACE1 antibody (1 mg/mL; Millipore, Billerica, MA); rabbit polyclonal BACE1 antibody and APP C-terminal antibody (500 μg/mL; EMD Biosciences, La Jolla, CA); APP N-terminus antibody (100 μg/mL, Millopore); Aβ17-24 antibody (1 mg/mL; 4G8, Covance) and Aβ1-12 antibody (BAM10, 500μg/mL; Sigma-Aldrich, St. Louis, MO); β-actin antibody (100 μg/mL; Sigma-Aldrich); rabbit anti-APP C-terminus polyclonal antibody (500 μg/mL; pAb369) generously provided by Dr. Sam Gandy; rabbit anti-APP C-terminus polyclonal antibody (pAb751/770, 100 μg/mL; Calbiochem); rabbit polyclonal oligomer/conformational (OC) antibody (500 μg/mL) was provided by Dr. Suhail Rasool and Dr. Charles G. Glabe at University of California.

    Techniques: In Vitro, Incubation, Centrifugation, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

    Immunoneutralization of endogenous sAPP-α promotes APP amyloidogenic processing. CHO/APP wt cells were treated with 2B3 or isotype-matched IgG 2b control antibody for 12 h as indicated. ( a ) Aβ species were analyzed in conditioned media from the treated CHO/APP wt cells by IB analysis using 6E10 antibody. ( b ) The Aβ ELISA results are represented as the mean ± SD of picograms of Aβ 40 or Aβ 42 per milligram of total intracellular protein after the 2B3 antibody treatment. In addition, these results are representative of three independent experiments with n = 3 for each condition. ( c ) Cell lysates were prepared and APP CTFs were analyzed by IB analysis using pAb751/770 (C-APP). ( d ) Densitometry shows the ratio (mean ± SD) of β-CTF to β-actin. For b , d , the results as presented are representative of three independent experiments with n = 3 for each condition. One-way ANOVA followed by post hoc comparison revealed a significant difference between the 2B3 antibody at either 2.5 or 5 μg/mL and isotype-matched IgG 2b control antibody treatment conditions. (*** P

    Journal: Nature communications

    Article Title: sAPP-? modulates ?-secretase activity and amyloid-? generation

    doi: 10.1038/ncomms1781

    Figure Lengend Snippet: Immunoneutralization of endogenous sAPP-α promotes APP amyloidogenic processing. CHO/APP wt cells were treated with 2B3 or isotype-matched IgG 2b control antibody for 12 h as indicated. ( a ) Aβ species were analyzed in conditioned media from the treated CHO/APP wt cells by IB analysis using 6E10 antibody. ( b ) The Aβ ELISA results are represented as the mean ± SD of picograms of Aβ 40 or Aβ 42 per milligram of total intracellular protein after the 2B3 antibody treatment. In addition, these results are representative of three independent experiments with n = 3 for each condition. ( c ) Cell lysates were prepared and APP CTFs were analyzed by IB analysis using pAb751/770 (C-APP). ( d ) Densitometry shows the ratio (mean ± SD) of β-CTF to β-actin. For b , d , the results as presented are representative of three independent experiments with n = 3 for each condition. One-way ANOVA followed by post hoc comparison revealed a significant difference between the 2B3 antibody at either 2.5 or 5 μg/mL and isotype-matched IgG 2b control antibody treatment conditions. (*** P

    Article Snippet: Other antibodies include: mouse monoclonal BACE1 antibody (1 mg/mL; Millipore, Billerica, MA); rabbit polyclonal BACE1 antibody and APP C-terminal antibody (500 μg/mL; EMD Biosciences, La Jolla, CA); APP N-terminus antibody (100 μg/mL, Millopore); Aβ17-24 antibody (1 mg/mL; 4G8, Covance) and Aβ1-12 antibody (BAM10, 500μg/mL; Sigma-Aldrich, St. Louis, MO); β-actin antibody (100 μg/mL; Sigma-Aldrich); rabbit anti-APP C-terminus polyclonal antibody (500 μg/mL; pAb369) generously provided by Dr. Sam Gandy; rabbit anti-APP C-terminus polyclonal antibody (pAb751/770, 100 μg/mL; Calbiochem); rabbit polyclonal oligomer/conformational (OC) antibody (500 μg/mL) was provided by Dr. Suhail Rasool and Dr. Charles G. Glabe at University of California.

    Techniques: Enzyme-linked Immunosorbent Assay

    sAPP-α inhibitsβ-secretase cleavage of APP in vitro . CHO cells expressing APP swe and wild-type human PS1 (CHO/APP swe /PS1 wt ) cells were treated with active (act.) or inactive (inact.) hsAPP-α recombinant protein at 0, 1, and 2 nM as indicated for 4 h. Secreted Aβ 40, 42 peptides in the cell culture medium were analyzed by ( a ) immunoblot (IB) and ( b ) Aβ ELISA analyses. The Aβ ELISA results are represented as the mean ± SD of picograms of Aβ 40 or Aβ 42 per milligram of total intracellular protein after hsAPP-α protein treatment. In addition, these results are representative of four independent experiments with n = 3 for each condition. ( c ) Cell lysates were prepared and APP CTFs were analyzed by IB using a rabbit polyclonal antibody against C-terminal APP (pAb751/770, C-APP). This β-CTF band was further confirmed by the additional IB using 6E10 antibody against Aβ 1-17 peptide for sAPP-α. ( d ) Relative ratio (mean ± SD) of β-CTF to β-actin was calculated by densitometry analysis. The results are representative of three independent experiments with n = 3 for each condition. One-way ANOVA followed by post hoc comparison revealed significant differences between 1 or 2 and 0 nM hsAPP-α protein treatment in both of Aβ 40, 42 reduction and relative ratio of β-CTF to β-actin. (*** P

    Journal: Nature communications

    Article Title: sAPP-? modulates ?-secretase activity and amyloid-? generation

    doi: 10.1038/ncomms1781

    Figure Lengend Snippet: sAPP-α inhibitsβ-secretase cleavage of APP in vitro . CHO cells expressing APP swe and wild-type human PS1 (CHO/APP swe /PS1 wt ) cells were treated with active (act.) or inactive (inact.) hsAPP-α recombinant protein at 0, 1, and 2 nM as indicated for 4 h. Secreted Aβ 40, 42 peptides in the cell culture medium were analyzed by ( a ) immunoblot (IB) and ( b ) Aβ ELISA analyses. The Aβ ELISA results are represented as the mean ± SD of picograms of Aβ 40 or Aβ 42 per milligram of total intracellular protein after hsAPP-α protein treatment. In addition, these results are representative of four independent experiments with n = 3 for each condition. ( c ) Cell lysates were prepared and APP CTFs were analyzed by IB using a rabbit polyclonal antibody against C-terminal APP (pAb751/770, C-APP). This β-CTF band was further confirmed by the additional IB using 6E10 antibody against Aβ 1-17 peptide for sAPP-α. ( d ) Relative ratio (mean ± SD) of β-CTF to β-actin was calculated by densitometry analysis. The results are representative of three independent experiments with n = 3 for each condition. One-way ANOVA followed by post hoc comparison revealed significant differences between 1 or 2 and 0 nM hsAPP-α protein treatment in both of Aβ 40, 42 reduction and relative ratio of β-CTF to β-actin. (*** P

    Article Snippet: Other antibodies include: mouse monoclonal BACE1 antibody (1 mg/mL; Millipore, Billerica, MA); rabbit polyclonal BACE1 antibody and APP C-terminal antibody (500 μg/mL; EMD Biosciences, La Jolla, CA); APP N-terminus antibody (100 μg/mL, Millopore); Aβ17-24 antibody (1 mg/mL; 4G8, Covance) and Aβ1-12 antibody (BAM10, 500μg/mL; Sigma-Aldrich, St. Louis, MO); β-actin antibody (100 μg/mL; Sigma-Aldrich); rabbit anti-APP C-terminus polyclonal antibody (500 μg/mL; pAb369) generously provided by Dr. Sam Gandy; rabbit anti-APP C-terminus polyclonal antibody (pAb751/770, 100 μg/mL; Calbiochem); rabbit polyclonal oligomer/conformational (OC) antibody (500 μg/mL) was provided by Dr. Suhail Rasool and Dr. Charles G. Glabe at University of California.

    Techniques: In Vitro, Expressing, Activated Clotting Time Assay, Recombinant, Cell Culture, Enzyme-linked Immunosorbent Assay

    Loss of Ei24 impairs glucose homeostasis. A , expression of Ei24 in islets from ob/ob mice, GK rats at 4 months of age, and C57BL/6 mice fed an HFD for 2 weeks and 8 weeks. β-Actin served as the loading control. CD , chow diet. B , total RNA was prepared from islets of WT and KO mice at 8 weeks of age. The transcription levels of Ei24 mRNA are normalized to β-actin mRNA. The results are representative of three individual experiments. C , Western blotting of Ei24 protein from isolated islets (300 islets/group) from WT and KO mice at 12 weeks of age. β-Actin served as the loading control. D , weight curves for WT and KO mice. The mean ± S.D. ( error bars ) of 15 mice is shown. E , concentration of the basic glucose curve for WT and KO mice. The mean ± S.D. of 10 mice is shown. F and G , glucose tolerance test ( GTT ) results of 10-week-old male ( F ) and female mice ( G ). Solid lines , WT mice ( n = 8); dashed lines , KO mice ( n = 8). H and I , ITT results of 10-week-old male ( H ) and female mice ( I ). Solid lines , WT mice ( n = 8); dashed lines , KO mice ( n = 8). J , in vivo GSIS detection in WT and KO mice. The results are representative of five replicates for each group. K , in vitro GSIS from isolated islets (70/group) of WT and KO mice by a fast digital perfusion system. The results are representative of three individual experiments. Data are expressed as the mean ± S.D. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Etoposide-induced protein 2.4 functions as a regulator of the calcium ATPase and protects pancreatic β-cell survival

    doi: 10.1074/jbc.RA118.002399

    Figure Lengend Snippet: Loss of Ei24 impairs glucose homeostasis. A , expression of Ei24 in islets from ob/ob mice, GK rats at 4 months of age, and C57BL/6 mice fed an HFD for 2 weeks and 8 weeks. β-Actin served as the loading control. CD , chow diet. B , total RNA was prepared from islets of WT and KO mice at 8 weeks of age. The transcription levels of Ei24 mRNA are normalized to β-actin mRNA. The results are representative of three individual experiments. C , Western blotting of Ei24 protein from isolated islets (300 islets/group) from WT and KO mice at 12 weeks of age. β-Actin served as the loading control. D , weight curves for WT and KO mice. The mean ± S.D. ( error bars ) of 15 mice is shown. E , concentration of the basic glucose curve for WT and KO mice. The mean ± S.D. of 10 mice is shown. F and G , glucose tolerance test ( GTT ) results of 10-week-old male ( F ) and female mice ( G ). Solid lines , WT mice ( n = 8); dashed lines , KO mice ( n = 8). H and I , ITT results of 10-week-old male ( H ) and female mice ( I ). Solid lines , WT mice ( n = 8); dashed lines , KO mice ( n = 8). J , in vivo GSIS detection in WT and KO mice. The results are representative of five replicates for each group. K , in vitro GSIS from isolated islets (70/group) of WT and KO mice by a fast digital perfusion system. The results are representative of three individual experiments. Data are expressed as the mean ± S.D. *, p

    Article Snippet: Antibodies against Ei24 (Sigma), proinsulin (Novus Biologicals), insulin (Abcam), c-PARP, total PARP (t-PARP), c-caspase-3, total caspase 3 (t-caspase-3), phosphorylated AMPK (p-AMPK), total AMPK (t-AMPK), p-ACC, total ACC (t-ACC), phosphorylated CAMKK2 (p-CAMKK2), ATP2a2, and β-actin (Cell Signaling Technology) were used, according to the manufacturer's protocols.

    Techniques: Expressing, Mouse Assay, Western Blot, Isolation, Concentration Assay, In Vivo, In Vitro

    Loss of Ei24 impairs AMPK activation and induces apoptotic cell death. A , ATP content of islets (40 islets/group), which were cultured in RPMI medium 1640 with 10% FBS from WT and KO mice at 8 weeks of age. Data were normalized to protein content. Data were obtained from three independent experiments. B , Western blotting for p-AMPK, t-AMPK, p-ACC, and t-ACC in the isolated islets (150 islets/group) from WT and KO mice at 8–10 weeks of age. C , Western blotting for p-CAMKK2 in the isolated islets (150 islets/group) from WT and KO mice at 8–10 weeks of age. D , Western blotting for p-AMPK, t-AMPK, c-PARP, t-PARP, c-caspase-3, and t-caspase-3 in the isolated islets (150 islets/group) with or without AICAR treatment from WT and KO mice at 8–10 weeks of age. β-Actin served as the loading control. Data were obtained from three independent experiments. Data are expressed as the mean ± S.D. ( error bars ): WT versus KO (*, p

    Journal: The Journal of Biological Chemistry

    Article Title: Etoposide-induced protein 2.4 functions as a regulator of the calcium ATPase and protects pancreatic β-cell survival

    doi: 10.1074/jbc.RA118.002399

    Figure Lengend Snippet: Loss of Ei24 impairs AMPK activation and induces apoptotic cell death. A , ATP content of islets (40 islets/group), which were cultured in RPMI medium 1640 with 10% FBS from WT and KO mice at 8 weeks of age. Data were normalized to protein content. Data were obtained from three independent experiments. B , Western blotting for p-AMPK, t-AMPK, p-ACC, and t-ACC in the isolated islets (150 islets/group) from WT and KO mice at 8–10 weeks of age. C , Western blotting for p-CAMKK2 in the isolated islets (150 islets/group) from WT and KO mice at 8–10 weeks of age. D , Western blotting for p-AMPK, t-AMPK, c-PARP, t-PARP, c-caspase-3, and t-caspase-3 in the isolated islets (150 islets/group) with or without AICAR treatment from WT and KO mice at 8–10 weeks of age. β-Actin served as the loading control. Data were obtained from three independent experiments. Data are expressed as the mean ± S.D. ( error bars ): WT versus KO (*, p

    Article Snippet: Antibodies against Ei24 (Sigma), proinsulin (Novus Biologicals), insulin (Abcam), c-PARP, total PARP (t-PARP), c-caspase-3, total caspase 3 (t-caspase-3), phosphorylated AMPK (p-AMPK), total AMPK (t-AMPK), p-ACC, total ACC (t-ACC), phosphorylated CAMKK2 (p-CAMKK2), ATP2a2, and β-actin (Cell Signaling Technology) were used, according to the manufacturer's protocols.

    Techniques: Activation Assay, Cell Culture, Mouse Assay, Western Blot, Isolation

    Loss of Ei24 causes apoptosis of pancreatic β cells. A , morphologies of islets. The islets from KO mice became uncompact and transparent compared with the WT islets. B , representative images of H E staining of islets from WT and KO mice at the age of 12 weeks. The degenerative β cells are indicated by black arrowheads. C , representative sections of islets stained for insulin ( red ) and nuclei ( blue ) from WT and KO mice at 12 weeks of age. Scale bars , 10 μm. D , density of β cells determined by counting the number of β cells in islets ( n = 40/group) from WT and KO mice. E , EM micrographs of WT and KO pancreatic β cells. The bottom panel in E shows enlargement of the boxed area in the top panel . The arrows indicate the dense core vesicles. F , the number of dense core vesicles ( DCV ) in pancreatic β cells was counted using Imaris software (cell numbers, n = 48/group). G , Western blotting for proinsulin in the isolated islets (30 islets/group) from WT and KO mice at 8 weeks of age. β-Actin served as the loading control. Data were obtained from three independent experiments. H , Western blotting for insulin in the isolated islets (60 islets/group) from WT and KO mice at 8 weeks of age. β-Actin served as the loading control. Data were obtained from three independent experiments. I , Western blotting for c-PARP, t-PARP, c-caspase-3, and t-caspase-3 in the isolated islets (150 islets/group) from WT and KO mice at 8–10 weeks of age. β-Actin served as the loading control. Data were obtained from five independent experiments. Data are expressed as the mean ± S.D. ( error bars ). *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Etoposide-induced protein 2.4 functions as a regulator of the calcium ATPase and protects pancreatic β-cell survival

    doi: 10.1074/jbc.RA118.002399

    Figure Lengend Snippet: Loss of Ei24 causes apoptosis of pancreatic β cells. A , morphologies of islets. The islets from KO mice became uncompact and transparent compared with the WT islets. B , representative images of H E staining of islets from WT and KO mice at the age of 12 weeks. The degenerative β cells are indicated by black arrowheads. C , representative sections of islets stained for insulin ( red ) and nuclei ( blue ) from WT and KO mice at 12 weeks of age. Scale bars , 10 μm. D , density of β cells determined by counting the number of β cells in islets ( n = 40/group) from WT and KO mice. E , EM micrographs of WT and KO pancreatic β cells. The bottom panel in E shows enlargement of the boxed area in the top panel . The arrows indicate the dense core vesicles. F , the number of dense core vesicles ( DCV ) in pancreatic β cells was counted using Imaris software (cell numbers, n = 48/group). G , Western blotting for proinsulin in the isolated islets (30 islets/group) from WT and KO mice at 8 weeks of age. β-Actin served as the loading control. Data were obtained from three independent experiments. H , Western blotting for insulin in the isolated islets (60 islets/group) from WT and KO mice at 8 weeks of age. β-Actin served as the loading control. Data were obtained from three independent experiments. I , Western blotting for c-PARP, t-PARP, c-caspase-3, and t-caspase-3 in the isolated islets (150 islets/group) from WT and KO mice at 8–10 weeks of age. β-Actin served as the loading control. Data were obtained from five independent experiments. Data are expressed as the mean ± S.D. ( error bars ). *, p

    Article Snippet: Antibodies against Ei24 (Sigma), proinsulin (Novus Biologicals), insulin (Abcam), c-PARP, total PARP (t-PARP), c-caspase-3, total caspase 3 (t-caspase-3), phosphorylated AMPK (p-AMPK), total AMPK (t-AMPK), p-ACC, total ACC (t-ACC), phosphorylated CAMKK2 (p-CAMKK2), ATP2a2, and β-actin (Cell Signaling Technology) were used, according to the manufacturer's protocols.

    Techniques: Mouse Assay, Staining, Software, Western Blot, Isolation

    EVI5 mediates the migration and invasion of NSCLC cells through the TGF-β/Smad signaling pathway. a Data obtained from several study groups deposited in the GEPIA2 database were analysed to explore the correlation between EVI5 and TGF-β receptors mRNA levels. b The levels of TGF-β receptor II, TGF-β receptor I, p-Smad3, Snail, N-cadherin, and MMP2 were significantly decreased and E-Cadherin was significantly increased in EVI5-knockdown cells compared with control cells. c Co-immunoprecipitation of EVI5 and TGF-β receptor II were shown, protein were immunoprecipitated from lysates of control and EVI5-overexpressing or EVI5-KO A549 cells using a specific monoclonal antibody. d EVI5-KO A549 and H226 cells treated or untreated with TGF-β1 (5 ng/ml) were allowed to migrate through 8-mm pore size transwell inserts. One day later, migratory and invasive cells were stained and counted in at least three light microscopic field, One representative image is shown (Cas-9 group compared with EVI5-KO group, Cas-9 + TGF-β1 group compared with EVI5-KO + TGF-β1 group). e EVI5-KO A549 cells were treated with TGF-β1 (5 ng/ml) for 24 h, and the expression levels of various proteins were then measured by western blot analysis. β-actin was used as the internal control. Bars represent mean ± SD from three independent experiments. Significant differences compared with the control:*** P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: EVI5 is an oncogene that regulates the proliferation and metastasis of NSCLC cells

    doi: 10.1186/s13046-020-01585-z

    Figure Lengend Snippet: EVI5 mediates the migration and invasion of NSCLC cells through the TGF-β/Smad signaling pathway. a Data obtained from several study groups deposited in the GEPIA2 database were analysed to explore the correlation between EVI5 and TGF-β receptors mRNA levels. b The levels of TGF-β receptor II, TGF-β receptor I, p-Smad3, Snail, N-cadherin, and MMP2 were significantly decreased and E-Cadherin was significantly increased in EVI5-knockdown cells compared with control cells. c Co-immunoprecipitation of EVI5 and TGF-β receptor II were shown, protein were immunoprecipitated from lysates of control and EVI5-overexpressing or EVI5-KO A549 cells using a specific monoclonal antibody. d EVI5-KO A549 and H226 cells treated or untreated with TGF-β1 (5 ng/ml) were allowed to migrate through 8-mm pore size transwell inserts. One day later, migratory and invasive cells were stained and counted in at least three light microscopic field, One representative image is shown (Cas-9 group compared with EVI5-KO group, Cas-9 + TGF-β1 group compared with EVI5-KO + TGF-β1 group). e EVI5-KO A549 cells were treated with TGF-β1 (5 ng/ml) for 24 h, and the expression levels of various proteins were then measured by western blot analysis. β-actin was used as the internal control. Bars represent mean ± SD from three independent experiments. Significant differences compared with the control:*** P

    Article Snippet: The following antibodies were used in the analysis: anti-EVI5 (Millipore, Billerica, MA, USA); anti-Emi1 and anti-TGF-β receptor II (Santa Cruz, CA, USA); anti-CyclinA2 (Proteintech, IL, USA); anti-pAkt (Ser473), anti-Akt, anti-Erk1/2, anti-pErk (Thr202/Tyr204), anti-CyclinD1, anti-MMP2, anti-p-Smad3, anti-Snail and anti-β-actin (Cell Signaling Technology, Danvers, MA, USA); anti-TGF-β receptor I (Abcam, London, UK); anti-N-cadherin and anti-Vimentin (BD Biosciences, USA); Anti-mouse and anti-rabbit secondary antibodies (Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Migration, Immunoprecipitation, Staining, Expressing, Western Blot

    MiR-486-5p suppresses EVI5 expression and reduces the proliferation, migration and invasion capability of NSCLC cells. a Schematic diagram showing the subcloning of a fragment containing the predicted miR-486-5p-binding site at positions 765–770 in the EVI5 3′-UTR into the psiCHECK-2 luciferase vector. Predicted duplex formation between miR-486-5p and the wild-type or mutant miR-486-5p-binding site is indicated. b-c Luciferase activity of the constructs containing the wild-type or the mutant EVI5 reporter gene in A549 and H226 cells co-transfected with miR-NC or miR-486-5p. A scrambled sequence was used as the NC. The relative Renilla luciferase activity was measured and normalized to firefly luciferase activity. d-e The expression of miR-486-5p and EVI5 in NSCLC cells transfected with miR-486-5p mimics was measured by qRT-PCR. f CCK-8 assay of cell viability in EVI5-overexpressing A549 cells transfected with miR-486-5p mimics or miR-NC. g Representative images of the clonogenic assay results for cell proliferation in EVI5-overexpressing A549 cells transfected with miR-486-5p mimics or miR-NC. h Representative images of the transwell assay results for cell migration and invasion in EVI5-overexpressing A549 cells transfected with miR-486-5p mimics or miR-NC. i EVI5-overexpressing A549 cells were transfected with miR-486-5p or miR-NC, and the expression levels of various proteins were then measured by western blot analysis. j The mRNA expression levels of miR-486-5p were measured by qRT-PCR and compared between 26 NSCLC and paired adjacent noncancerous lung tissues. k qRT-PCR analysis of relative miR-486-5p expression levels in human NSCLC cell lines. β-actin was used as the internal control. Bars represent mean ± SD from three independent experiments. Significant differences compared with the control: * P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: EVI5 is an oncogene that regulates the proliferation and metastasis of NSCLC cells

    doi: 10.1186/s13046-020-01585-z

    Figure Lengend Snippet: MiR-486-5p suppresses EVI5 expression and reduces the proliferation, migration and invasion capability of NSCLC cells. a Schematic diagram showing the subcloning of a fragment containing the predicted miR-486-5p-binding site at positions 765–770 in the EVI5 3′-UTR into the psiCHECK-2 luciferase vector. Predicted duplex formation between miR-486-5p and the wild-type or mutant miR-486-5p-binding site is indicated. b-c Luciferase activity of the constructs containing the wild-type or the mutant EVI5 reporter gene in A549 and H226 cells co-transfected with miR-NC or miR-486-5p. A scrambled sequence was used as the NC. The relative Renilla luciferase activity was measured and normalized to firefly luciferase activity. d-e The expression of miR-486-5p and EVI5 in NSCLC cells transfected with miR-486-5p mimics was measured by qRT-PCR. f CCK-8 assay of cell viability in EVI5-overexpressing A549 cells transfected with miR-486-5p mimics or miR-NC. g Representative images of the clonogenic assay results for cell proliferation in EVI5-overexpressing A549 cells transfected with miR-486-5p mimics or miR-NC. h Representative images of the transwell assay results for cell migration and invasion in EVI5-overexpressing A549 cells transfected with miR-486-5p mimics or miR-NC. i EVI5-overexpressing A549 cells were transfected with miR-486-5p or miR-NC, and the expression levels of various proteins were then measured by western blot analysis. j The mRNA expression levels of miR-486-5p were measured by qRT-PCR and compared between 26 NSCLC and paired adjacent noncancerous lung tissues. k qRT-PCR analysis of relative miR-486-5p expression levels in human NSCLC cell lines. β-actin was used as the internal control. Bars represent mean ± SD from three independent experiments. Significant differences compared with the control: * P

    Article Snippet: The following antibodies were used in the analysis: anti-EVI5 (Millipore, Billerica, MA, USA); anti-Emi1 and anti-TGF-β receptor II (Santa Cruz, CA, USA); anti-CyclinA2 (Proteintech, IL, USA); anti-pAkt (Ser473), anti-Akt, anti-Erk1/2, anti-pErk (Thr202/Tyr204), anti-CyclinD1, anti-MMP2, anti-p-Smad3, anti-Snail and anti-β-actin (Cell Signaling Technology, Danvers, MA, USA); anti-TGF-β receptor I (Abcam, London, UK); anti-N-cadherin and anti-Vimentin (BD Biosciences, USA); Anti-mouse and anti-rabbit secondary antibodies (Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Expressing, Migration, Subcloning, Binding Assay, Luciferase, Plasmid Preparation, Mutagenesis, Activity Assay, Construct, Transfection, Sequencing, Quantitative RT-PCR, CCK-8 Assay, Clonogenic Assay, Transwell Assay, Western Blot

    Inhibition of NSCLC cell proliferation, migration and invasion by overexpressing miR-486-5p. a CCK-8 assay of cell viability in NSCLC cell lines transfected with miR-486-5p mimics at 24, 48, and 72 h. b Representative images of the clonogenic assay results for NSCLC cell proliferation. c Flow cytometric analysis of A549 and H226 cells (cells transfected with miR-486-5p mimics vs. miR-NC). Cells were harvested 72 h after transfection and stained with Annexin V/FITC and PI. d Wound healing assay was performed to evaluate the effect of miR-486-5p transfection on cells. e Representative images of the transwell assay results for cell migration and invasion in A549 and H226 cells transfected with miR-486-5p mimics or miR-NC. f A549 and H226 cells were treated with or without miR-486-5p mimics for 72 h. The levels of Emi1, p-Akt, Akt, p-Erk, Erk, CyclinA2, Cyclin D1, TGF-β receptor II, TGF-β receptor I, p-Smad3, Snail, E-cadherin and N-cadherin were analysed by western blotting. β-actin was used as the internal control. Bars represent mean ± SD from three independent experiments. Significant differences compared with the control: ** P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: EVI5 is an oncogene that regulates the proliferation and metastasis of NSCLC cells

    doi: 10.1186/s13046-020-01585-z

    Figure Lengend Snippet: Inhibition of NSCLC cell proliferation, migration and invasion by overexpressing miR-486-5p. a CCK-8 assay of cell viability in NSCLC cell lines transfected with miR-486-5p mimics at 24, 48, and 72 h. b Representative images of the clonogenic assay results for NSCLC cell proliferation. c Flow cytometric analysis of A549 and H226 cells (cells transfected with miR-486-5p mimics vs. miR-NC). Cells were harvested 72 h after transfection and stained with Annexin V/FITC and PI. d Wound healing assay was performed to evaluate the effect of miR-486-5p transfection on cells. e Representative images of the transwell assay results for cell migration and invasion in A549 and H226 cells transfected with miR-486-5p mimics or miR-NC. f A549 and H226 cells were treated with or without miR-486-5p mimics for 72 h. The levels of Emi1, p-Akt, Akt, p-Erk, Erk, CyclinA2, Cyclin D1, TGF-β receptor II, TGF-β receptor I, p-Smad3, Snail, E-cadherin and N-cadherin were analysed by western blotting. β-actin was used as the internal control. Bars represent mean ± SD from three independent experiments. Significant differences compared with the control: ** P

    Article Snippet: The following antibodies were used in the analysis: anti-EVI5 (Millipore, Billerica, MA, USA); anti-Emi1 and anti-TGF-β receptor II (Santa Cruz, CA, USA); anti-CyclinA2 (Proteintech, IL, USA); anti-pAkt (Ser473), anti-Akt, anti-Erk1/2, anti-pErk (Thr202/Tyr204), anti-CyclinD1, anti-MMP2, anti-p-Smad3, anti-Snail and anti-β-actin (Cell Signaling Technology, Danvers, MA, USA); anti-TGF-β receptor I (Abcam, London, UK); anti-N-cadherin and anti-Vimentin (BD Biosciences, USA); Anti-mouse and anti-rabbit secondary antibodies (Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Inhibition, Migration, CCK-8 Assay, Transfection, Clonogenic Assay, Staining, Wound Healing Assay, Transwell Assay, Western Blot

    EVI5 is upregulated in NSCLC tissues and cell lines. a-b Data obtained from several study groups deposited in the Oncomine database ( http://www.oncomine.org ) were analysed to compare the differences in EVI5 expression between lung cancer and normal lung tissue. c EVI5 mRNA levels in 40 NSCLC tissues and paired oncancerous lung tissues. d Relative mRNA expression levels of EVI5 in 40 paired NSCLC tissues. The Y axis indicates the log10 transformed fold change in the T/N protein expression ratios of EVI5. The number of each specimen is indicated below the X axis. e Effect of the EVI5 mRNA expression level on overall survival in 1926 NSCLC patients. Kaplan–Meier plots were generated using Kaplan–Meier Plotter ( http://www.kmplot.com ). f Total RNA and protein were extracted from several cell lines, and the expression of EVI5 at the mRNA and protein levels was measured by qRT-PCR and western blotting, respectively. β-actin was used as the internal control. Bars represent mean ± SD from three independent experiments. Significant differences compared with the control: * P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: EVI5 is an oncogene that regulates the proliferation and metastasis of NSCLC cells

    doi: 10.1186/s13046-020-01585-z

    Figure Lengend Snippet: EVI5 is upregulated in NSCLC tissues and cell lines. a-b Data obtained from several study groups deposited in the Oncomine database ( http://www.oncomine.org ) were analysed to compare the differences in EVI5 expression between lung cancer and normal lung tissue. c EVI5 mRNA levels in 40 NSCLC tissues and paired oncancerous lung tissues. d Relative mRNA expression levels of EVI5 in 40 paired NSCLC tissues. The Y axis indicates the log10 transformed fold change in the T/N protein expression ratios of EVI5. The number of each specimen is indicated below the X axis. e Effect of the EVI5 mRNA expression level on overall survival in 1926 NSCLC patients. Kaplan–Meier plots were generated using Kaplan–Meier Plotter ( http://www.kmplot.com ). f Total RNA and protein were extracted from several cell lines, and the expression of EVI5 at the mRNA and protein levels was measured by qRT-PCR and western blotting, respectively. β-actin was used as the internal control. Bars represent mean ± SD from three independent experiments. Significant differences compared with the control: * P

    Article Snippet: The following antibodies were used in the analysis: anti-EVI5 (Millipore, Billerica, MA, USA); anti-Emi1 and anti-TGF-β receptor II (Santa Cruz, CA, USA); anti-CyclinA2 (Proteintech, IL, USA); anti-pAkt (Ser473), anti-Akt, anti-Erk1/2, anti-pErk (Thr202/Tyr204), anti-CyclinD1, anti-MMP2, anti-p-Smad3, anti-Snail and anti-β-actin (Cell Signaling Technology, Danvers, MA, USA); anti-TGF-β receptor I (Abcam, London, UK); anti-N-cadherin and anti-Vimentin (BD Biosciences, USA); Anti-mouse and anti-rabbit secondary antibodies (Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Expressing, Transformation Assay, Generated, Quantitative RT-PCR, Western Blot

    Knockdown of EVI5 inhibits cell proliferation, migration and invasion in vitro. a EVI5 mRNA and protein levels in EVI5-knockdown NSCLC cells. b CCK-8 assay of cell viability in A549 and H226 cells (si-EVI5 compared with si-NC). c Flow cytometry assay of A549 and H226 cells (si-EVI5 compared with si-NC). Cells were harvested 72 h after transfection and stained with PI. The percentage of cells in each cell cycle phase is shown in the inset of each panel. d Wound healing assay was performed to evaluate cell migration in A549 and H226 cells (si-EVI5 compared with si-NC). e Representative images of the transwell assay results for cell migration and invasion in A549 and H226 cells (si-EVI5 compared with si-NC). β-actin was used as the internal control. Bars represent mean ± SD from three independent experiments. Significant differences compared with the control: * P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: EVI5 is an oncogene that regulates the proliferation and metastasis of NSCLC cells

    doi: 10.1186/s13046-020-01585-z

    Figure Lengend Snippet: Knockdown of EVI5 inhibits cell proliferation, migration and invasion in vitro. a EVI5 mRNA and protein levels in EVI5-knockdown NSCLC cells. b CCK-8 assay of cell viability in A549 and H226 cells (si-EVI5 compared with si-NC). c Flow cytometry assay of A549 and H226 cells (si-EVI5 compared with si-NC). Cells were harvested 72 h after transfection and stained with PI. The percentage of cells in each cell cycle phase is shown in the inset of each panel. d Wound healing assay was performed to evaluate cell migration in A549 and H226 cells (si-EVI5 compared with si-NC). e Representative images of the transwell assay results for cell migration and invasion in A549 and H226 cells (si-EVI5 compared with si-NC). β-actin was used as the internal control. Bars represent mean ± SD from three independent experiments. Significant differences compared with the control: * P

    Article Snippet: The following antibodies were used in the analysis: anti-EVI5 (Millipore, Billerica, MA, USA); anti-Emi1 and anti-TGF-β receptor II (Santa Cruz, CA, USA); anti-CyclinA2 (Proteintech, IL, USA); anti-pAkt (Ser473), anti-Akt, anti-Erk1/2, anti-pErk (Thr202/Tyr204), anti-CyclinD1, anti-MMP2, anti-p-Smad3, anti-Snail and anti-β-actin (Cell Signaling Technology, Danvers, MA, USA); anti-TGF-β receptor I (Abcam, London, UK); anti-N-cadherin and anti-Vimentin (BD Biosciences, USA); Anti-mouse and anti-rabbit secondary antibodies (Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Migration, In Vitro, CCK-8 Assay, Flow Cytometry, Transfection, Staining, Wound Healing Assay, Transwell Assay

    Effects of NSF in a DSS-induced colitis mouse model. (A, B) Mice were orally treated with KRG (200 mg/kg), NSF (200 mg/kg), or SF (200 mg/kg) once a day along with 3% DSS in tap water for seven days. After sacrifice of the mice, colon lengths were measured using a ruler. (C) MPO activity as an indicator of neutrophil infiltration in the stomach was analyzed in total lysates of colon. (D) Body weight increase was determined by changes in body weight after oral administration of NSF. (E) Colon weight per length ratio was calculated by measured colon weight and length scales. (F) Protein levels of COX-1, ZO-1, occludin, and β-actin in the colons of DSS-induced colitis mice treated with NSF were determined by immunoblotting. # p

    Journal: Journal of Ginseng Research

    Article Title: Gastroprotective effects of the nonsaponin fraction of Korean Red Ginseng through cyclooxygenase-1 upregulation

    doi: 10.1016/j.jgr.2019.11.001

    Figure Lengend Snippet: Effects of NSF in a DSS-induced colitis mouse model. (A, B) Mice were orally treated with KRG (200 mg/kg), NSF (200 mg/kg), or SF (200 mg/kg) once a day along with 3% DSS in tap water for seven days. After sacrifice of the mice, colon lengths were measured using a ruler. (C) MPO activity as an indicator of neutrophil infiltration in the stomach was analyzed in total lysates of colon. (D) Body weight increase was determined by changes in body weight after oral administration of NSF. (E) Colon weight per length ratio was calculated by measured colon weight and length scales. (F) Protein levels of COX-1, ZO-1, occludin, and β-actin in the colons of DSS-induced colitis mice treated with NSF were determined by immunoblotting. # p

    Article Snippet: COX-1 and β-actin antibodies were purchased from Cell Signaling Technology (Beverly, MA).

    Techniques: Mouse Assay, Activity Assay

    Effects of NSF on cell viability and COX-1 expression in RAW264.7 cells. (A) RAW264.7 cells were treated with NSF (0-200 μg/ml) for 24 h, and cell viability was determined by MTT assay. (B) RAW264.7 cells were treated with NSF (0-200 μg/ml) for 24 h, and the mRNA levels of COX-1 and GAPDH were determined by RT-PCR. (C) Levels of COX-1 and β-actin in whole cell lysates of RAW264.7 cells treated with NSF were determined by immunoblotting. Band intensity was measured by ImageJ. COX-1, cyclooxygenase-1; GAPDH, glyceradehyde-3-phosphate dehydrogenase; NSF, nonsaponin fraction; RT-PCR, reverse transcription polymerase chain reaction.

    Journal: Journal of Ginseng Research

    Article Title: Gastroprotective effects of the nonsaponin fraction of Korean Red Ginseng through cyclooxygenase-1 upregulation

    doi: 10.1016/j.jgr.2019.11.001

    Figure Lengend Snippet: Effects of NSF on cell viability and COX-1 expression in RAW264.7 cells. (A) RAW264.7 cells were treated with NSF (0-200 μg/ml) for 24 h, and cell viability was determined by MTT assay. (B) RAW264.7 cells were treated with NSF (0-200 μg/ml) for 24 h, and the mRNA levels of COX-1 and GAPDH were determined by RT-PCR. (C) Levels of COX-1 and β-actin in whole cell lysates of RAW264.7 cells treated with NSF were determined by immunoblotting. Band intensity was measured by ImageJ. COX-1, cyclooxygenase-1; GAPDH, glyceradehyde-3-phosphate dehydrogenase; NSF, nonsaponin fraction; RT-PCR, reverse transcription polymerase chain reaction.

    Article Snippet: COX-1 and β-actin antibodies were purchased from Cell Signaling Technology (Beverly, MA).

    Techniques: Expressing, MTT Assay, Reverse Transcription Polymerase Chain Reaction

    Effects of NSF in an indomethacin-induced gastritis rat model. (A, B) SD rats were orally treated with NSF (100 or 200 mg/kg) or omeprazole (10 mg/kg) once a day along with indomethacin for five days. After sacrifice of the rats, gastric lesions were imaged using an optical digital camera. Formation of stomach lesions was evaluated using a pixel counter. (C) The pH of collected gastric juice was measured by a pH meter. (D, E) Histological examination of sections of gastric tissue stained with hematoxylin and eosin. Images were captured using an optical digital camera. Thicknesses of gastric walls were measured using ImageJ. (F) COX-1 mRNA level was determined by real-time PCR. (G) Protein levels of COX-1 and β-actin were determined by immunoblotting. (H) MPO activity as an indicator of neutrophil infiltration in the stomach was analyzed in total lysates of stomach. # p

    Journal: Journal of Ginseng Research

    Article Title: Gastroprotective effects of the nonsaponin fraction of Korean Red Ginseng through cyclooxygenase-1 upregulation

    doi: 10.1016/j.jgr.2019.11.001

    Figure Lengend Snippet: Effects of NSF in an indomethacin-induced gastritis rat model. (A, B) SD rats were orally treated with NSF (100 or 200 mg/kg) or omeprazole (10 mg/kg) once a day along with indomethacin for five days. After sacrifice of the rats, gastric lesions were imaged using an optical digital camera. Formation of stomach lesions was evaluated using a pixel counter. (C) The pH of collected gastric juice was measured by a pH meter. (D, E) Histological examination of sections of gastric tissue stained with hematoxylin and eosin. Images were captured using an optical digital camera. Thicknesses of gastric walls were measured using ImageJ. (F) COX-1 mRNA level was determined by real-time PCR. (G) Protein levels of COX-1 and β-actin were determined by immunoblotting. (H) MPO activity as an indicator of neutrophil infiltration in the stomach was analyzed in total lysates of stomach. # p

    Article Snippet: COX-1 and β-actin antibodies were purchased from Cell Signaling Technology (Beverly, MA).

    Techniques: Staining, Real-time Polymerase Chain Reaction, Activity Assay

    The effect of BV and melittin on the expression of apoptosis regulatory proteins in A375SM melanoma cells. Cells were treated with BV and melittin for 24 h, and the expression levels of cleaved caspase-3 and cleaved caspase-9 were detected by Western blotting. The levels of β-actin were used as an internal control. Each value represents the mean ± SE from three independent experiments.

    Journal: Molecules

    Article Title: Bee Venom and Its Peptide Component Melittin Suppress Growth and Migration of Melanoma Cells via Inhibition of PI3K/AKT/mTOR and MAPK Pathways

    doi: 10.3390/molecules24050929

    Figure Lengend Snippet: The effect of BV and melittin on the expression of apoptosis regulatory proteins in A375SM melanoma cells. Cells were treated with BV and melittin for 24 h, and the expression levels of cleaved caspase-3 and cleaved caspase-9 were detected by Western blotting. The levels of β-actin were used as an internal control. Each value represents the mean ± SE from three independent experiments.

    Article Snippet: Anti-phospho-PI3K, anti-PI3K, anti-phospho-AKT, anti-AKT, anti-phospho-mTOR, anti-mTOR, anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-p38, anti-p38, anti-cleaved caspase-3, anti-cleaved capase-9, anti-MITF, anti-MMP-2, anti-MMP-9 and anti-β-actin antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Expressing, Western Blot

    The effect of BV and melittin on the regulation of PI3K/AKT/mTOR and MAPK pathways. A375SM melanoma cells were treated with ( A ) BV, melittin and ( B ) MG132, and the protein levels were detected by Western blot analysis using specific antibodies. The levels of β-actin were used as an internal control. Each value represents the mean ± SE from three independent experiments.

    Journal: Molecules

    Article Title: Bee Venom and Its Peptide Component Melittin Suppress Growth and Migration of Melanoma Cells via Inhibition of PI3K/AKT/mTOR and MAPK Pathways

    doi: 10.3390/molecules24050929

    Figure Lengend Snippet: The effect of BV and melittin on the regulation of PI3K/AKT/mTOR and MAPK pathways. A375SM melanoma cells were treated with ( A ) BV, melittin and ( B ) MG132, and the protein levels were detected by Western blot analysis using specific antibodies. The levels of β-actin were used as an internal control. Each value represents the mean ± SE from three independent experiments.

    Article Snippet: Anti-phospho-PI3K, anti-PI3K, anti-phospho-AKT, anti-AKT, anti-phospho-mTOR, anti-mTOR, anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-p38, anti-p38, anti-cleaved caspase-3, anti-cleaved capase-9, anti-MITF, anti-MMP-2, anti-MMP-9 and anti-β-actin antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Western Blot

    Knockdown of AQP5 suppressed the ability of migration and invasion in HCT116 and SW480 cells. a The ability of migration was measured by scratch wound healing assay after knockdown of AQP5, the migration rate was determined. b The invasiveness of HCT116 and SW480 cells with and without AQP5 silencing was evaluated by transwell assay, invasive cells were counted and expressed as fold change compared to the Mock group. c The expression levels of MMP-2 and MMP-9 were detected by immunoblotting after knockdown of AQP5, the bands were semi-quantified by densitometry, normalized to β-actin expression and expressed as fold change compared with the Mock group. d The activities of MMP-2 and MMP-9 in AQP5-silenced and control cells were assessed by gelatin zymography. Data were presented as the mean ± SD. * p

    Journal: Cytotechnology

    Article Title: Anti-cancer effect of Aquaporin 5 silencing in colorectal cancer cells in association with inhibition of Wnt/β-catenin pathway

    doi: 10.1007/s10616-017-0147-7

    Figure Lengend Snippet: Knockdown of AQP5 suppressed the ability of migration and invasion in HCT116 and SW480 cells. a The ability of migration was measured by scratch wound healing assay after knockdown of AQP5, the migration rate was determined. b The invasiveness of HCT116 and SW480 cells with and without AQP5 silencing was evaluated by transwell assay, invasive cells were counted and expressed as fold change compared to the Mock group. c The expression levels of MMP-2 and MMP-9 were detected by immunoblotting after knockdown of AQP5, the bands were semi-quantified by densitometry, normalized to β-actin expression and expressed as fold change compared with the Mock group. d The activities of MMP-2 and MMP-9 in AQP5-silenced and control cells were assessed by gelatin zymography. Data were presented as the mean ± SD. * p

    Article Snippet: Proteins from the cell lysates were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), transferred onto Polyvinylidene Fluoride (PVDF) membranes, and immunoblotted respectively with antibodies against AQP5 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-514022), β-catenin (BOSTER Biological Technology, Pleasanton, CA, USA, BA0426), MMP2 (BOSTER, BA0569), MMP9 (BOSTER, BA0573), E-cadherin (BOSTER, BA0474), Vimentin (Bioss Antibodies, Woburn, MA, USA, bs-8533R), N-cadherin (BOSTER, BA0673), uPA (Bioss, bs-1927R), TIMP-1 (Bioss, bs-0415R), TIMP-2 (Bioss, bs-10395R), Snail (Bioss, bs-1371R), Wnt1 (BOSTER, BA3158-2), β-actin (Santa Cruz Biotechnology, sc-47778).

    Techniques: Migration, Wound Healing Assay, Transwell Assay, Expressing, Zymography

    Silencing of AQP5 inhibited Wnt/β-catenin signal transduction. a The expression levels of Wnt1 and β-catenin were detected by western blotting after silencing AQP5 in HCT116 and SW480 cells. b Following transfection of β-catenin S33Y in AQP5-silenced HCT116 and SW480 cells, the expression of β-catenin, E-cadherin, Vimentin, N-cadherin and Snail was determined by western blotting. The bands were semi-quantified by densitometry, normalized to β-actin expression and expressed as fold change compared with the Mock group. Data were presented as the mean ± SD. * p

    Journal: Cytotechnology

    Article Title: Anti-cancer effect of Aquaporin 5 silencing in colorectal cancer cells in association with inhibition of Wnt/β-catenin pathway

    doi: 10.1007/s10616-017-0147-7

    Figure Lengend Snippet: Silencing of AQP5 inhibited Wnt/β-catenin signal transduction. a The expression levels of Wnt1 and β-catenin were detected by western blotting after silencing AQP5 in HCT116 and SW480 cells. b Following transfection of β-catenin S33Y in AQP5-silenced HCT116 and SW480 cells, the expression of β-catenin, E-cadherin, Vimentin, N-cadherin and Snail was determined by western blotting. The bands were semi-quantified by densitometry, normalized to β-actin expression and expressed as fold change compared with the Mock group. Data were presented as the mean ± SD. * p

    Article Snippet: Proteins from the cell lysates were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), transferred onto Polyvinylidene Fluoride (PVDF) membranes, and immunoblotted respectively with antibodies against AQP5 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-514022), β-catenin (BOSTER Biological Technology, Pleasanton, CA, USA, BA0426), MMP2 (BOSTER, BA0569), MMP9 (BOSTER, BA0573), E-cadherin (BOSTER, BA0474), Vimentin (Bioss Antibodies, Woburn, MA, USA, bs-8533R), N-cadherin (BOSTER, BA0673), uPA (Bioss, bs-1927R), TIMP-1 (Bioss, bs-0415R), TIMP-2 (Bioss, bs-10395R), Snail (Bioss, bs-1371R), Wnt1 (BOSTER, BA3158-2), β-actin (Santa Cruz Biotechnology, sc-47778).

    Techniques: Transduction, Expressing, Western Blot, Transfection

    Knockdown of AQP5 regulated the expression of EMT-related proteins in HCT116 and SW480 cells. a The expression levels of E-cadherin, Vimentin, N-cadherin, uPA, TIMP-1, TIMP-2 and Snail were measured by western-blot analysis after knockdown of AQP5, the bands were semi-quantified by densitometry, normalized to β-actin expression and expressed as fold change compared with the Mock group. b Immunofluorescence staining was performed to detect the expression of E-cadherin and Snail in HCT116 and SW480 cells with and without AQP5 silencing. Data were presented as the mean ± SD. * p

    Journal: Cytotechnology

    Article Title: Anti-cancer effect of Aquaporin 5 silencing in colorectal cancer cells in association with inhibition of Wnt/β-catenin pathway

    doi: 10.1007/s10616-017-0147-7

    Figure Lengend Snippet: Knockdown of AQP5 regulated the expression of EMT-related proteins in HCT116 and SW480 cells. a The expression levels of E-cadherin, Vimentin, N-cadherin, uPA, TIMP-1, TIMP-2 and Snail were measured by western-blot analysis after knockdown of AQP5, the bands were semi-quantified by densitometry, normalized to β-actin expression and expressed as fold change compared with the Mock group. b Immunofluorescence staining was performed to detect the expression of E-cadherin and Snail in HCT116 and SW480 cells with and without AQP5 silencing. Data were presented as the mean ± SD. * p

    Article Snippet: Proteins from the cell lysates were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), transferred onto Polyvinylidene Fluoride (PVDF) membranes, and immunoblotted respectively with antibodies against AQP5 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-514022), β-catenin (BOSTER Biological Technology, Pleasanton, CA, USA, BA0426), MMP2 (BOSTER, BA0569), MMP9 (BOSTER, BA0573), E-cadherin (BOSTER, BA0474), Vimentin (Bioss Antibodies, Woburn, MA, USA, bs-8533R), N-cadherin (BOSTER, BA0673), uPA (Bioss, bs-1927R), TIMP-1 (Bioss, bs-0415R), TIMP-2 (Bioss, bs-10395R), Snail (Bioss, bs-1371R), Wnt1 (BOSTER, BA3158-2), β-actin (Santa Cruz Biotechnology, sc-47778).

    Techniques: Expressing, Western Blot, Immunofluorescence, Staining

    Knockdown of AQP5 in HCT116 and SW480 cells. a The expression of AQP5 protein was assessed by western-blot assay after knockdown of AQP5, the bands were semi-quantified by densitometry, normalized to β-actin expression and expressed as fold change compared with the Mock group. b The expression of AQP5 mRNA was measured by real-time PCR after silencing AQP5. Data were presented as the mean ± SD. ** p

    Journal: Cytotechnology

    Article Title: Anti-cancer effect of Aquaporin 5 silencing in colorectal cancer cells in association with inhibition of Wnt/β-catenin pathway

    doi: 10.1007/s10616-017-0147-7

    Figure Lengend Snippet: Knockdown of AQP5 in HCT116 and SW480 cells. a The expression of AQP5 protein was assessed by western-blot assay after knockdown of AQP5, the bands were semi-quantified by densitometry, normalized to β-actin expression and expressed as fold change compared with the Mock group. b The expression of AQP5 mRNA was measured by real-time PCR after silencing AQP5. Data were presented as the mean ± SD. ** p

    Article Snippet: Proteins from the cell lysates were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), transferred onto Polyvinylidene Fluoride (PVDF) membranes, and immunoblotted respectively with antibodies against AQP5 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-514022), β-catenin (BOSTER Biological Technology, Pleasanton, CA, USA, BA0426), MMP2 (BOSTER, BA0569), MMP9 (BOSTER, BA0573), E-cadherin (BOSTER, BA0474), Vimentin (Bioss Antibodies, Woburn, MA, USA, bs-8533R), N-cadherin (BOSTER, BA0673), uPA (Bioss, bs-1927R), TIMP-1 (Bioss, bs-0415R), TIMP-2 (Bioss, bs-10395R), Snail (Bioss, bs-1371R), Wnt1 (BOSTER, BA3158-2), β-actin (Santa Cruz Biotechnology, sc-47778).

    Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction

    Pulmonary muscarinic receptors function and expression in allergic AIRmin and AIRmax mice. AIRmin or AIRmax mice were sensitized and challenged with OVA as in  Figure 4 . Control group consisted of nonmanipulated animals. The experiments were performed 24 h after the last OVA challenge. Respiratory pattern of allergic AIRmin (a and c) or AIRmax (b and d) to inhaled MCh was evaluated in the presence or absence of gallamine. Penh values were used as an index of bronchoconstriction induced after sequential delivery of increasing concentrations of MCh. Area under the curve was obtained from Penh values (c and d). Gene (e and g) or protein (f and h) expression of the M2 and M3 muscarinic receptors was evaluated by real-time PCR or Western blot analysis in lungs from OVA-sensitized AIRmin and AIRmax mice. The real-time PCR was carried out using  β -actin gene expression as internal control for normalization of M3R (e) and M2R (g) mRNA transcription levels. In Western blot analysis the density of M3R (f) and M2R (h) protein expression was normalized to actin expression in each sample. Data are expressed as mean ± SEM of four mice per group and are representative of two experiments. Western blots data were quantified by densitometry using the ImageJ software (NIH). Statistical analyses of Student's  t  test for (a), (b), (e), (f), (g), and (h). Statistical analyses of ANOVA following Tukey HSD for (c) and (d). * P

    Journal: BioMed Research International

    Article Title: Role of M2 Muscarinic Receptor in the Airway Response to Methacholine of Mice Selected for Minimal or Maximal Acute Inflammatory Response

    doi: 10.1155/2013/805627

    Figure Lengend Snippet: Pulmonary muscarinic receptors function and expression in allergic AIRmin and AIRmax mice. AIRmin or AIRmax mice were sensitized and challenged with OVA as in Figure 4 . Control group consisted of nonmanipulated animals. The experiments were performed 24 h after the last OVA challenge. Respiratory pattern of allergic AIRmin (a and c) or AIRmax (b and d) to inhaled MCh was evaluated in the presence or absence of gallamine. Penh values were used as an index of bronchoconstriction induced after sequential delivery of increasing concentrations of MCh. Area under the curve was obtained from Penh values (c and d). Gene (e and g) or protein (f and h) expression of the M2 and M3 muscarinic receptors was evaluated by real-time PCR or Western blot analysis in lungs from OVA-sensitized AIRmin and AIRmax mice. The real-time PCR was carried out using β -actin gene expression as internal control for normalization of M3R (e) and M2R (g) mRNA transcription levels. In Western blot analysis the density of M3R (f) and M2R (h) protein expression was normalized to actin expression in each sample. Data are expressed as mean ± SEM of four mice per group and are representative of two experiments. Western blots data were quantified by densitometry using the ImageJ software (NIH). Statistical analyses of Student's t test for (a), (b), (e), (f), (g), and (h). Statistical analyses of ANOVA following Tukey HSD for (c) and (d). * P

    Article Snippet: The following polyclonal rabbit antisera were used: anti-M1 (1 : 200), anti-M2 (1 : 100), anti-M3 (1 : 100), and anti-β -actin (1 : 400) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). β -actin protein expression was used as an internal standard for relative quantification of muscarinic receptors expression levels.

    Techniques: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Western Blot, Software

    Respiratory pattern and expression of muscarinic receptors in AIRmin and AIRmax mice. Respiratory pattern was determined in awake, unrestrained mice by noninvasive whole-body barometric plethysmography. (a) Penh values were used as an index of bronchoconstriction induced after sequential delivery of increasing concentrations of MCh (3, 6, 12, and 25 mg/mL) and (b) provocative concentration of aerosol MCh at a 200% increase (PC200) over baseline values. Gene (c and e) or protein (d and f) expression of M2 and M3 muscarinic receptors was evaluated by real-time PCR or Western blot analysis in lungs from AIRmin and AIRmax mice. Real-time PCR was carried out using  β -actin gene expression as internal control for normalization of M3R (c) and M2R (e) mRNA transcription levels. All PCR reactions were quantitative reactions made by real-time PCR. In Western blot analysis the density of M3R (d) and M2R (f) protein expression was nor aerosol at a malized to actin expression in each sample. Western blots were quantified by densitometry using the ImageJ software (NIH). Real-time PCR and Western-blot analyses were performed using pooled lungs from 5 mice. Data are expressed as mean ± SEM of five mice per group and are representative of three experiments; * P

    Journal: BioMed Research International

    Article Title: Role of M2 Muscarinic Receptor in the Airway Response to Methacholine of Mice Selected for Minimal or Maximal Acute Inflammatory Response

    doi: 10.1155/2013/805627

    Figure Lengend Snippet: Respiratory pattern and expression of muscarinic receptors in AIRmin and AIRmax mice. Respiratory pattern was determined in awake, unrestrained mice by noninvasive whole-body barometric plethysmography. (a) Penh values were used as an index of bronchoconstriction induced after sequential delivery of increasing concentrations of MCh (3, 6, 12, and 25 mg/mL) and (b) provocative concentration of aerosol MCh at a 200% increase (PC200) over baseline values. Gene (c and e) or protein (d and f) expression of M2 and M3 muscarinic receptors was evaluated by real-time PCR or Western blot analysis in lungs from AIRmin and AIRmax mice. Real-time PCR was carried out using β -actin gene expression as internal control for normalization of M3R (c) and M2R (e) mRNA transcription levels. All PCR reactions were quantitative reactions made by real-time PCR. In Western blot analysis the density of M3R (d) and M2R (f) protein expression was nor aerosol at a malized to actin expression in each sample. Western blots were quantified by densitometry using the ImageJ software (NIH). Real-time PCR and Western-blot analyses were performed using pooled lungs from 5 mice. Data are expressed as mean ± SEM of five mice per group and are representative of three experiments; * P

    Article Snippet: The following polyclonal rabbit antisera were used: anti-M1 (1 : 200), anti-M2 (1 : 100), anti-M3 (1 : 100), and anti-β -actin (1 : 400) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). β -actin protein expression was used as an internal standard for relative quantification of muscarinic receptors expression levels.

    Techniques: Expressing, Mouse Assay, Concentration Assay, Real-time Polymerase Chain Reaction, Western Blot, Polymerase Chain Reaction, Software

    HCV infection affects intracellular localization, expression levels, and activities of focal adhesion molecules. ( A ) Immunofluorescence of paxillin ( green ), and alpha-actinin ( red ) by confocal microscopy in Ctrl and HCV Huh7.5.1 cells after 24 hrs from plating. DAPI ( blue ) was included to stain the nuclei. Magnification bar: 30 µ. ( B ) Paxillin and alpha-actinin protein expression levels observed 24 hrs after plating. ( C ) Immunohistochemical analysis of paxillin expression in HCCs and control livers (×200 magnification). ( D ) Total FAK and tyrosine 397 phosphorylated FAK were observed 24 hrs after plating. Immunoblots are representative of at least four independent experiments. ( E ) Immunohistochemical analysis of tyrosine 397 phosphorylated FAK expression in HCCs and control livers (×200 magnification). In left histograms densitometric analysis is reported as fold changes in protein levels respect to the control cells considered as 1 after normalization against beta-actin (as loading control). *P

    Journal: PLoS ONE

    Article Title: Focal Adhesion Kinase (FAK) Mediates the Induction of Pro-Oncogenic and Fibrogenic Phenotypes in Hepatitis C Virus (HCV)-Infected Cells

    doi: 10.1371/journal.pone.0044147

    Figure Lengend Snippet: HCV infection affects intracellular localization, expression levels, and activities of focal adhesion molecules. ( A ) Immunofluorescence of paxillin ( green ), and alpha-actinin ( red ) by confocal microscopy in Ctrl and HCV Huh7.5.1 cells after 24 hrs from plating. DAPI ( blue ) was included to stain the nuclei. Magnification bar: 30 µ. ( B ) Paxillin and alpha-actinin protein expression levels observed 24 hrs after plating. ( C ) Immunohistochemical analysis of paxillin expression in HCCs and control livers (×200 magnification). ( D ) Total FAK and tyrosine 397 phosphorylated FAK were observed 24 hrs after plating. Immunoblots are representative of at least four independent experiments. ( E ) Immunohistochemical analysis of tyrosine 397 phosphorylated FAK expression in HCCs and control livers (×200 magnification). In left histograms densitometric analysis is reported as fold changes in protein levels respect to the control cells considered as 1 after normalization against beta-actin (as loading control). *P

    Article Snippet: Antibodies Antibodies used: anti-core protein monoclonal antibody (Affinity BioReagents, Inc., Golden, CO); anti-NS3, anti-FAK, anti-paxillin, anti-alpha-actinin, anti-alpha-smooth muscle actin, anti-beta-actin and anti-phosphotyrosine monoclonal antibodies (Santa Cruz Biotech.); peroxidase-conjugated goat anti rabbit and anti-mouse IgG (Sigma-Aldrich Inc.); FITC-conjugated anti-mouse and TRIC-conjugated anti-rabbit (Sigma-Aldrich Inc.).

    Techniques: Infection, Expressing, Immunofluorescence, Confocal Microscopy, Staining, Immunohistochemistry, Western Blot

    HCV affects proliferation, anchorage-independent growth, adhesion and migration of Huh7.5.1 cells. ( A ) RT-PCR for the 5′UTR in control (Ctrl) and HCV-infected Huh7.5.1 cells (HCV). ( B ) Protein expression levels of HCV core ( upper panel ), HCV NS3 ( middle panel ) and beta-actin (loading control, lower panel ) in control (Ctrl) and HCV-infected Huh7.5.1 cells (HCV). Immunoblots are representative of at least five independent experiments. ( C ) Proliferation rate was evaluated as incorporation of BrdU performed at three different time points: 0, 6 and 24 hrs. Quantitative data of the analysis of BrdU incorporation was converted in unit of induction with respect to the Ctrl considered as 1. Histograms are the mean value ±SD ( bars ) of five independent experiments. *P

    Journal: PLoS ONE

    Article Title: Focal Adhesion Kinase (FAK) Mediates the Induction of Pro-Oncogenic and Fibrogenic Phenotypes in Hepatitis C Virus (HCV)-Infected Cells

    doi: 10.1371/journal.pone.0044147

    Figure Lengend Snippet: HCV affects proliferation, anchorage-independent growth, adhesion and migration of Huh7.5.1 cells. ( A ) RT-PCR for the 5′UTR in control (Ctrl) and HCV-infected Huh7.5.1 cells (HCV). ( B ) Protein expression levels of HCV core ( upper panel ), HCV NS3 ( middle panel ) and beta-actin (loading control, lower panel ) in control (Ctrl) and HCV-infected Huh7.5.1 cells (HCV). Immunoblots are representative of at least five independent experiments. ( C ) Proliferation rate was evaluated as incorporation of BrdU performed at three different time points: 0, 6 and 24 hrs. Quantitative data of the analysis of BrdU incorporation was converted in unit of induction with respect to the Ctrl considered as 1. Histograms are the mean value ±SD ( bars ) of five independent experiments. *P

    Article Snippet: Antibodies Antibodies used: anti-core protein monoclonal antibody (Affinity BioReagents, Inc., Golden, CO); anti-NS3, anti-FAK, anti-paxillin, anti-alpha-actinin, anti-alpha-smooth muscle actin, anti-beta-actin and anti-phosphotyrosine monoclonal antibodies (Santa Cruz Biotech.); peroxidase-conjugated goat anti rabbit and anti-mouse IgG (Sigma-Aldrich Inc.); FITC-conjugated anti-mouse and TRIC-conjugated anti-rabbit (Sigma-Aldrich Inc.).

    Techniques: Migration, Reverse Transcription Polymerase Chain Reaction, Infection, Expressing, Western Blot, BrdU Incorporation Assay

    The combination of berberine with resveratrol increased LDLR expression in HepG2 cells. Cells were cultured in 6-well plate for 24 h with a 3 × 10 5 cell density, and then culture medium were replaced by fresh medium containing different concentration of FBS indicated in A, or 1% FBS and drugs indicated in B for another 24 h followed by extraction of the total proteins from the cells. A : the effect of different concentration FBS on LDLR expression were analyzed by western blot assay. Then the band intensity was quantified by grey scanning analysis, and the intensity ratio of LDLR to β-actin in 0% FBS group was set to 1. *** p

    Journal: International Journal of Molecular Sciences

    Article Title: Combination of Berberine with Resveratrol Improves the Lipid-Lowering Efficacy

    doi: 10.3390/ijms19123903

    Figure Lengend Snippet: The combination of berberine with resveratrol increased LDLR expression in HepG2 cells. Cells were cultured in 6-well plate for 24 h with a 3 × 10 5 cell density, and then culture medium were replaced by fresh medium containing different concentration of FBS indicated in A, or 1% FBS and drugs indicated in B for another 24 h followed by extraction of the total proteins from the cells. A : the effect of different concentration FBS on LDLR expression were analyzed by western blot assay. Then the band intensity was quantified by grey scanning analysis, and the intensity ratio of LDLR to β-actin in 0% FBS group was set to 1. *** p

    Article Snippet: Goat antibodies directed against LDLR (C-7, sc-18823), HRP-labeled anti-goat IgG (sc-2354) and anti-β-actin antibody (sc-8432) were from Santa Cruz Biotechnology (Heidelberg, German).

    Techniques: Expressing, Cell Culture, Concentration Assay, Western Blot

    NanoATV and HIV-1 endosomal protein regulation. Western blot of Rab5, −7, −11, LAMP1 and β-actin was performed in cell lysates from MDM treated with native ATV or nanoATV and infected with HIV-1 at day 0, 5 or 10 post-drug treatment then incubated for 7 days. Uninfected cells and infected cells without drug treatment served as negative and positive controls for differential expression of cellular proteins during HIV-1 infection. Blots shown are from one donor and experiment, and equivalent to two independent experiments performed.

    Journal: Retrovirology

    Article Title: Opposing regulation of endolysosomal pathways by long-acting nanoformulated antiretroviral therapy and HIV-1 in human macrophages

    doi: 10.1186/s12977-014-0133-5

    Figure Lengend Snippet: NanoATV and HIV-1 endosomal protein regulation. Western blot of Rab5, −7, −11, LAMP1 and β-actin was performed in cell lysates from MDM treated with native ATV or nanoATV and infected with HIV-1 at day 0, 5 or 10 post-drug treatment then incubated for 7 days. Uninfected cells and infected cells without drug treatment served as negative and positive controls for differential expression of cellular proteins during HIV-1 infection. Blots shown are from one donor and experiment, and equivalent to two independent experiments performed.

    Article Snippet: Rabbit anti-human Rab 5, −7, −11, LAMP1 and β-actin antibodies were purchased from Santa Cruz Biotechnology, Dallas, TX, USA.

    Techniques: Western Blot, Infection, Incubation, Expressing

    Nuclear import of the fusion protein GST-GFP-NLS in the permeabilized cells. Cells permeabilized with 30 µg/mL digitonin (Dig30) and non-permeabilized cells (Control) were incubated with GST-GFP-NLS in the absence (−Extract) or presence (+Extract) of Xenopus egg extract supplemented with ATP for 1 h at 25 °C. The incorporation of GST-GFP-NLS was assessed by green fluorescence. Cell nuclei were counterstained with Hoechst 33243. Note that without egg extract, the nuclei are not labelled (arrows). Due to light scattering of the fluorescence in the cytoplasm, the nuclei appear smaller than they are. The egg extract restored nuclear import in the permeabilized cells. These pictures are representative of five independent replicates. Scale bar = 20 µm. Detection of importin alpha-1 (55 kDa) ( B ) and Karyopherin beta -1 (KPNB1–97 kDa) ( C ) in Xenopus egg extract by western blot in the presence (+) or in absence (−) of the anti- Xenopus importin alpha-1 antibody (clone 15) and the anti-rat KPNB1 antibody (KPNB1-clone 23). Beta actin was used as a loading control. M: size markers. The cropped blots came from the same gels and were analyzed with the same exposure times (B:1 sec; C:1 min).

    Journal: Scientific Reports

    Article Title: Nuclear import of Xenopus egg extract components into cultured cells for reprogramming purposes: a case study on goldfish fin cells

    doi: 10.1038/s41598-019-39500-y

    Figure Lengend Snippet: Nuclear import of the fusion protein GST-GFP-NLS in the permeabilized cells. Cells permeabilized with 30 µg/mL digitonin (Dig30) and non-permeabilized cells (Control) were incubated with GST-GFP-NLS in the absence (−Extract) or presence (+Extract) of Xenopus egg extract supplemented with ATP for 1 h at 25 °C. The incorporation of GST-GFP-NLS was assessed by green fluorescence. Cell nuclei were counterstained with Hoechst 33243. Note that without egg extract, the nuclei are not labelled (arrows). Due to light scattering of the fluorescence in the cytoplasm, the nuclei appear smaller than they are. The egg extract restored nuclear import in the permeabilized cells. These pictures are representative of five independent replicates. Scale bar = 20 µm. Detection of importin alpha-1 (55 kDa) ( B ) and Karyopherin beta -1 (KPNB1–97 kDa) ( C ) in Xenopus egg extract by western blot in the presence (+) or in absence (−) of the anti- Xenopus importin alpha-1 antibody (clone 15) and the anti-rat KPNB1 antibody (KPNB1-clone 23). Beta actin was used as a loading control. M: size markers. The cropped blots came from the same gels and were analyzed with the same exposure times (B:1 sec; C:1 min).

    Article Snippet: After western blotting of the egg extract, the membranes were incubated overnight at 4 °C with rabbit polyclonal Xenopus anti-Lamin B3 (1:10 000, a gift from N. Morin, France), Xenopus anti-importin alpha1 (1:5000, a gift from K. Weiss, Switzerland), mouse monoclonal rat anti-karyopherin β1 (1:1000 KPNB1, Antibodies-online, clone 23), and anti-beta actin (1:5000, Sigma, clone AC15).

    Techniques: Incubation, Fluorescence, Western Blot, Size-exclusion Chromatography

    Incorporation of Lamin B3 from Xenopus eggs into the nuclei of permeabilized cells. ( A ) Detection of Lamin B3 (68 kDa) in Xenopus egg extract (Extract) by western blot in the presence (+) or absence (−) of anti- Xenopus Lamin B3. Beta actin was used as a loading control. M: size markers. The cropped blots came from the same gel and were analyzed with the same exposure times (Lamin B3:2 sec; beta actin: 1 min). ( B ) Nuclear import of Lamin B3 detected by immunofluorescence in permeabilized (Dig30) and non-permeabilized (Control) cells incubated for 60 min in the presence (+Extract) of Xenopus egg extract under energy supplementation at 25 °C. Lamin B3-positive cells presenting a detectable signal (from weak to strong staining) were observed. A high proportion of nuclei were labelled. Inset: magnification of one nucleus showing a strong labelling of the nuclear lamina. These pictures are representative of five independent replicates. Scale bar = 20 µm.

    Journal: Scientific Reports

    Article Title: Nuclear import of Xenopus egg extract components into cultured cells for reprogramming purposes: a case study on goldfish fin cells

    doi: 10.1038/s41598-019-39500-y

    Figure Lengend Snippet: Incorporation of Lamin B3 from Xenopus eggs into the nuclei of permeabilized cells. ( A ) Detection of Lamin B3 (68 kDa) in Xenopus egg extract (Extract) by western blot in the presence (+) or absence (−) of anti- Xenopus Lamin B3. Beta actin was used as a loading control. M: size markers. The cropped blots came from the same gel and were analyzed with the same exposure times (Lamin B3:2 sec; beta actin: 1 min). ( B ) Nuclear import of Lamin B3 detected by immunofluorescence in permeabilized (Dig30) and non-permeabilized (Control) cells incubated for 60 min in the presence (+Extract) of Xenopus egg extract under energy supplementation at 25 °C. Lamin B3-positive cells presenting a detectable signal (from weak to strong staining) were observed. A high proportion of nuclei were labelled. Inset: magnification of one nucleus showing a strong labelling of the nuclear lamina. These pictures are representative of five independent replicates. Scale bar = 20 µm.

    Article Snippet: After western blotting of the egg extract, the membranes were incubated overnight at 4 °C with rabbit polyclonal Xenopus anti-Lamin B3 (1:10 000, a gift from N. Morin, France), Xenopus anti-importin alpha1 (1:5000, a gift from K. Weiss, Switzerland), mouse monoclonal rat anti-karyopherin β1 (1:1000 KPNB1, Antibodies-online, clone 23), and anti-beta actin (1:5000, Sigma, clone AC15).

    Techniques: Western Blot, Size-exclusion Chromatography, Immunofluorescence, Incubation, Staining

    a Expression levels of TSPO measured in protein extracts from rat and murine glioma cell lines (9L, C6 and GL261). Western blots were normalized using the anti-β-actin antibody. H E staining ( b ) and immunohistochemistry for TSPO ( c ) of coronal brain sections illustrating tumour growth and high level of TSPO expression in a 9L glioma

    Journal: European Journal of Nuclear Medicine and Molecular Imaging

    Article Title: The translocator protein ligand [18F]DPA-714 images glioma and activated microglia in vivo

    doi: 10.1007/s00259-011-2041-4

    Figure Lengend Snippet: a Expression levels of TSPO measured in protein extracts from rat and murine glioma cell lines (9L, C6 and GL261). Western blots were normalized using the anti-β-actin antibody. H E staining ( b ) and immunohistochemistry for TSPO ( c ) of coronal brain sections illustrating tumour growth and high level of TSPO expression in a 9L glioma

    Article Snippet: Incubation with the primary antibodies diluted in PBST with 3% of BSA for the anti-TSPO antibody and 3% non-fat dry milk for the anti-β-actin antibody, respectively (1/10,000 dilution of the anti-rat TSPO antibody NP155 [ ] generously provided by Dr. M. Higuchi, NIRS, Japan, and 1/5,000 dilution of the anti-β-actin antibody purchased from Sigma Aldrich), was performed overnight at 4°C.

    Techniques: Expressing, Western Blot, Staining, Immunohistochemistry

    BACE1 Western blot. (A) Representative Western blot of BACE1 (top images) and beta-actin (bottom images) in MFC (left) and MTC (right). APOE ε3/3 carriers showed higher BACE1 signals than APOE ε4 heterozygotes and homozygotes in ND samples.

    Journal: Current Alzheimer research

    Article Title: BACE1 Levels by APOE Genotype in Non-Demented and Alzheimer\u2019s Post-Mortem Brains

    doi:

    Figure Lengend Snippet: BACE1 Western blot. (A) Representative Western blot of BACE1 (top images) and beta-actin (bottom images) in MFC (left) and MTC (right). APOE ε3/3 carriers showed higher BACE1 signals than APOE ε4 heterozygotes and homozygotes in ND samples.

    Article Snippet: The membranes were then stripped and reprobed with mouse anti-β actin (#A1978; Sigma-Aldrich, St. Louis, MO).

    Techniques: Western Blot

    Activation of Wnt signal induces midbrain characteristics in human ESC-derived NPCs. a Efficient induction of neural rosette cells from human ESCs by co-treatment with dorsomorphin and SB431542. b Strong immunoreactivity for SOX1 and Nestin in neural rosette cells. Morphology of neural rosette cells expanding in either the absence ( c ) or the presence of 1 μM BIO ( d ). e NPCs treated with BIO maintained immunoreactivity for SOX1 and Nestin. f Treatment with BIO upregulated EN1 expression and downregulated expressions of BF1 and GBX2 in dose-dependent manner. g BIO treatment significantly increased the number of EN1-positive neural cells. h The inductive effect of BIO treatment on midbrain fate appeared to be more specific than that of FGF8. i Expression pattern of another set of regional markers ( SIX3 for forebrain; PAX2 for midbrain; and HOXA2 for hindbrain) supported the midbrain-biased fate of NPCs treated with BIO. Treatment with other known GSK3 inhibitors, 1-AKP ( j ) and LiCl ( k ), resulted in regionalization comparable to BIO treatment. l Treating NPCs with Wnt antagonists (100 ng/ml DKK-1 and 500 ng/ml frizzled-5) downregulated the endogenous level of EN1 transcript in NPCs. m Immunoblot for β-catenin and EN1 protein after introduction of two different β-catenin-specific shRNAs (shRNA-1 and shRNA-2). EN1 protein level was directly downregulated by β-catenin knockdown. β-actin was a loading control. All data are expressed as mean ± S.E.M. Statistical significance was estimated using Student’s t test ( g , i , j , and k ) or one-way ANOVA ( f , h , and l ) from at least three independent experiments; * p

    Journal: Experimental & Molecular Medicine

    Article Title: Wnt signal activation induces midbrain specification through direct binding of the beta-catenin/TCF4 complex to the EN1 promoter in human pluripotent stem cells

    doi: 10.1038/s12276-018-0044-y

    Figure Lengend Snippet: Activation of Wnt signal induces midbrain characteristics in human ESC-derived NPCs. a Efficient induction of neural rosette cells from human ESCs by co-treatment with dorsomorphin and SB431542. b Strong immunoreactivity for SOX1 and Nestin in neural rosette cells. Morphology of neural rosette cells expanding in either the absence ( c ) or the presence of 1 μM BIO ( d ). e NPCs treated with BIO maintained immunoreactivity for SOX1 and Nestin. f Treatment with BIO upregulated EN1 expression and downregulated expressions of BF1 and GBX2 in dose-dependent manner. g BIO treatment significantly increased the number of EN1-positive neural cells. h The inductive effect of BIO treatment on midbrain fate appeared to be more specific than that of FGF8. i Expression pattern of another set of regional markers ( SIX3 for forebrain; PAX2 for midbrain; and HOXA2 for hindbrain) supported the midbrain-biased fate of NPCs treated with BIO. Treatment with other known GSK3 inhibitors, 1-AKP ( j ) and LiCl ( k ), resulted in regionalization comparable to BIO treatment. l Treating NPCs with Wnt antagonists (100 ng/ml DKK-1 and 500 ng/ml frizzled-5) downregulated the endogenous level of EN1 transcript in NPCs. m Immunoblot for β-catenin and EN1 protein after introduction of two different β-catenin-specific shRNAs (shRNA-1 and shRNA-2). EN1 protein level was directly downregulated by β-catenin knockdown. β-actin was a loading control. All data are expressed as mean ± S.E.M. Statistical significance was estimated using Student’s t test ( g , i , j , and k ) or one-way ANOVA ( f , h , and l ) from at least three independent experiments; * p

    Article Snippet: After incubation with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) for 1 h at room temperature, the membrane was incubated with primary antibodies (mouse anti-β-catenin (Santa Cruz Biotechnology), mouse anti-EN1 (Abcam, Cambridge, UK), and mouse anti-β-actin (Sigma-Aldrich)) for 1 h at room temperature or overnight at 4 °C.

    Techniques: Activation Assay, Derivative Assay, Expressing, ALP Assay, shRNA

    Differential expression of A3A mRNA in progenitor as compared to non-progenitor CBMCs. (A) Six CBMC samples were sorted into progenitor and non-progenitor cells, and A3A mRNA expression was assessed and normalized to GAPDH mRNA level. A3A expression in six pairs of samples was on average 228 (SD = 298) fold higher in non-progenitors as compared to progenitors, corresponding to differential G-to-A editing in these populations. (B) Three pairs of pooled CBMC samples were sorted into progenitor and non-progenitor subpopulations and examined for A3A level, showing higher levels in non-progenitors. (C) Quantitation of the A3A Western blot bands normalized to beta-Actin, confirming higher A3A levels in non-progenitor cells.

    Journal: PLoS ONE

    Article Title: APOBEC3A Is Implicated in a Novel Class of G-to-A mRNA Editing in WT1 Transcripts

    doi: 10.1371/journal.pone.0120089

    Figure Lengend Snippet: Differential expression of A3A mRNA in progenitor as compared to non-progenitor CBMCs. (A) Six CBMC samples were sorted into progenitor and non-progenitor cells, and A3A mRNA expression was assessed and normalized to GAPDH mRNA level. A3A expression in six pairs of samples was on average 228 (SD = 298) fold higher in non-progenitors as compared to progenitors, corresponding to differential G-to-A editing in these populations. (B) Three pairs of pooled CBMC samples were sorted into progenitor and non-progenitor subpopulations and examined for A3A level, showing higher levels in non-progenitors. (C) Quantitation of the A3A Western blot bands normalized to beta-Actin, confirming higher A3A levels in non-progenitor cells.

    Article Snippet: Protein was detected using rabbit anti-APOBEC3A (Santa Cruz Biotechnology) and mouse anti-beta-Actin (Sigma-Aldrich), followed by HRP-conjugated goat anti-rabbit and anti-mouse secondary antibodies (Sigma-Aldrich), respectively.

    Techniques: Expressing, Quantitation Assay, Western Blot

    TET1-CD up-regulates TSGs expression. (A). The SMMC 7721 cells were transiently transfected with either TET1-CD plasmids or TET1-mCD plasmids. Expression of TET1-CD and TET1-mCD proteins was analysed by Western blot, and β-actin was used as an internal control. (B) (C). The SMMC 7721 cells were cultured in the 6 mm plate for 24h and then transiently transfected with either pflag-CMV4 vector (control), TET1-CD or TET1-mCD. Expression of TSGs and oncogenes was analyzed by Quantitative RT-PCR 48h after transient transfection, and the results were represented as mean ± SD of three independent experiments. *p

    Journal: PLoS ONE

    Article Title: Effects of a single transient transfection of Ten-eleven translocation 1 catalytic domain on hepatocellular carcinoma

    doi: 10.1371/journal.pone.0207139

    Figure Lengend Snippet: TET1-CD up-regulates TSGs expression. (A). The SMMC 7721 cells were transiently transfected with either TET1-CD plasmids or TET1-mCD plasmids. Expression of TET1-CD and TET1-mCD proteins was analysed by Western blot, and β-actin was used as an internal control. (B) (C). The SMMC 7721 cells were cultured in the 6 mm plate for 24h and then transiently transfected with either pflag-CMV4 vector (control), TET1-CD or TET1-mCD. Expression of TSGs and oncogenes was analyzed by Quantitative RT-PCR 48h after transient transfection, and the results were represented as mean ± SD of three independent experiments. *p

    Article Snippet: Then, the membrane was blocked with 5% non-fat milk for 2h at room temperature, and then incubated at 4°C overnight with anti-TET1 (GeneTex) and anti-FLAG (Stratagene) antibody (1:1000 dilution), or mouse monoclonal anti-β-actin (Sigma) antibody (1:5000 dilution) for the internal control.

    Techniques: Expressing, Transfection, Western Blot, Cell Culture, Plasmid Preparation, Quantitative RT-PCR

    Blimp1 promotes lung cancer cell migration and is aberrantly expressed in multiple cancers. (A) A549 cells or (B) H441 cells were transiently transfected with 1 µg of Blimp1 cDNA or EV DNA using Lipofectamine 2000. Upper panels: WCE were isolated after 48 h and subjected to immunoblot analysis for Blimp1 and β-actin. Lower panels: Alternatively, 24 h after transfection, cells were subjected to a migration assay as in Fig. 1 . The average migration from three independent experiments ± SD is presented relative to the EV (set at 1.0). P values were calculated using a Student's t -test. *, P

    Journal: PLoS ONE

    Article Title: Blimp1 Activation by AP-1 in Human Lung Cancer Cells Promotes a Migratory Phenotype and Is Inhibited by the Lysyl Oxidase Propeptide

    doi: 10.1371/journal.pone.0033287

    Figure Lengend Snippet: Blimp1 promotes lung cancer cell migration and is aberrantly expressed in multiple cancers. (A) A549 cells or (B) H441 cells were transiently transfected with 1 µg of Blimp1 cDNA or EV DNA using Lipofectamine 2000. Upper panels: WCE were isolated after 48 h and subjected to immunoblot analysis for Blimp1 and β-actin. Lower panels: Alternatively, 24 h after transfection, cells were subjected to a migration assay as in Fig. 1 . The average migration from three independent experiments ± SD is presented relative to the EV (set at 1.0). P values were calculated using a Student's t -test. *, P

    Article Snippet: Antibodies against β-actin (AC-15) and α-tubulin (DM1A) were from Sigma.

    Techniques: Migration, Transfection, Isolation

    A Ras to c-Raf pathway induces the Blimp1 promoter and AP-1 activity. (A) A549 cells were transfected with 5 µg of a plasmid expressing dominant negative Ras S186 or EV DNA. After 48 h, WCE and RNA were prepared. Samples (30 µg) of WCE were subjected to immunoblot analysis for Blimp1, Ras and α-tubulin. The bands were quantified using NIH Image J software and Blimp1 expression normalized to β-actin expression. The average values for normalized Blimp1 levels from two independent experiments are given relative to EV DNA (set to 1.0). (B) RNA was isolated from the A549 cells treated as in part A, and subjected to Q-PCR for BLIMP1 mRNA and normalized to GAPDH . The values represent an average of two independent experiments. (C) A549 cells were transfected, in triplicate, with 0.16 µg of Ras S186 plasmid or EV DNA, 0.33 µg of a MSV- β-gal expression vector and 0.16 µg of the 7-kB Blimp1 promoter Blimp1 -Luc, in a 12-well plate. After 48 h, cell lysates were subjected to measurements for luciferase and β-gal activities and normalized Blimp1 promoter activity values are presented as the mean ± SEM from two experiments (EV DNA set to 1.0). (D) Two-hundred pmol of an siRNA against K-Ras or a negative control siRNA (Ctrl) was incubated in the presence of 25 µl of Lipofectamine RNAiMAX in 2 ml of optiMEM in P100 plates. A549 cells (6.4×10 5 ) were seeded at a final siRNA concentration of 20 nM for 48 h. WCE were subjected to immunoblotting for K-Ras, Blimp1, c-Jun, phospho-ERK (p-ERK), Fra-1, Fra-2, and α-tubulin. Average normalized levels of Blimp1, c-Jun, Fra-1, Fra-2 and K-Ras from two independent experiments are given relative to the control (set to 1.0). Immunoblots from one of two independent experiments with similar results are presented. (E) Two-hundred pmol of an siRNA against c- RAF or a negative control siRNA was incubated in the presence of 25 µl of Lipofectamine RNAiMAX in 2 ml of optiMEM in P100 plates. A549 cells (6.4×10 5 ) were seeded at a final siRNA concentration of 20 nM for 48 h. WCE were subjected to immunoblotting for c-Raf, Blimp1, Fra-1, Fra-2, c-Jun, and α-tubulin. Average normalized levels of c-Raf, Blimp1, Fra-1, Fra-2 and c-Jun from two independent experiments are given relative to the control (set to 1.0). Immunoblots from one of two independent experiments with similar results are presented. (F) A549 cells were transiently transfected, in triplicate, with si-c-RAF or negative control siRNA at a final concentration of 20 nM in a 12-well plate. Eight h later, Blimp1 -luc promoter construct (0.16 µg) and an MSV- β-gal expression vector (0.33 µg) were transfected into these siRNA-treated A549 cells for an additional 40 h. Relative (Rel.) Blimp1 promoter activity values are presented as the mean ± SEM from two experiments (EV DNA set to 1.0).

    Journal: PLoS ONE

    Article Title: Blimp1 Activation by AP-1 in Human Lung Cancer Cells Promotes a Migratory Phenotype and Is Inhibited by the Lysyl Oxidase Propeptide

    doi: 10.1371/journal.pone.0033287

    Figure Lengend Snippet: A Ras to c-Raf pathway induces the Blimp1 promoter and AP-1 activity. (A) A549 cells were transfected with 5 µg of a plasmid expressing dominant negative Ras S186 or EV DNA. After 48 h, WCE and RNA were prepared. Samples (30 µg) of WCE were subjected to immunoblot analysis for Blimp1, Ras and α-tubulin. The bands were quantified using NIH Image J software and Blimp1 expression normalized to β-actin expression. The average values for normalized Blimp1 levels from two independent experiments are given relative to EV DNA (set to 1.0). (B) RNA was isolated from the A549 cells treated as in part A, and subjected to Q-PCR for BLIMP1 mRNA and normalized to GAPDH . The values represent an average of two independent experiments. (C) A549 cells were transfected, in triplicate, with 0.16 µg of Ras S186 plasmid or EV DNA, 0.33 µg of a MSV- β-gal expression vector and 0.16 µg of the 7-kB Blimp1 promoter Blimp1 -Luc, in a 12-well plate. After 48 h, cell lysates were subjected to measurements for luciferase and β-gal activities and normalized Blimp1 promoter activity values are presented as the mean ± SEM from two experiments (EV DNA set to 1.0). (D) Two-hundred pmol of an siRNA against K-Ras or a negative control siRNA (Ctrl) was incubated in the presence of 25 µl of Lipofectamine RNAiMAX in 2 ml of optiMEM in P100 plates. A549 cells (6.4×10 5 ) were seeded at a final siRNA concentration of 20 nM for 48 h. WCE were subjected to immunoblotting for K-Ras, Blimp1, c-Jun, phospho-ERK (p-ERK), Fra-1, Fra-2, and α-tubulin. Average normalized levels of Blimp1, c-Jun, Fra-1, Fra-2 and K-Ras from two independent experiments are given relative to the control (set to 1.0). Immunoblots from one of two independent experiments with similar results are presented. (E) Two-hundred pmol of an siRNA against c- RAF or a negative control siRNA was incubated in the presence of 25 µl of Lipofectamine RNAiMAX in 2 ml of optiMEM in P100 plates. A549 cells (6.4×10 5 ) were seeded at a final siRNA concentration of 20 nM for 48 h. WCE were subjected to immunoblotting for c-Raf, Blimp1, Fra-1, Fra-2, c-Jun, and α-tubulin. Average normalized levels of c-Raf, Blimp1, Fra-1, Fra-2 and c-Jun from two independent experiments are given relative to the control (set to 1.0). Immunoblots from one of two independent experiments with similar results are presented. (F) A549 cells were transiently transfected, in triplicate, with si-c-RAF or negative control siRNA at a final concentration of 20 nM in a 12-well plate. Eight h later, Blimp1 -luc promoter construct (0.16 µg) and an MSV- β-gal expression vector (0.33 µg) were transfected into these siRNA-treated A549 cells for an additional 40 h. Relative (Rel.) Blimp1 promoter activity values are presented as the mean ± SEM from two experiments (EV DNA set to 1.0).

    Article Snippet: Antibodies against β-actin (AC-15) and α-tubulin (DM1A) were from Sigma.

    Techniques: Activity Assay, Transfection, Plasmid Preparation, Expressing, Dominant Negative Mutation, Software, Isolation, Polymerase Chain Reaction, Luciferase, Negative Control, Incubation, Concentration Assay, Western Blot, Construct

    Blimp1 is expressed in lung cancer cells and its knockdown reduces migration. (A) Samples of nuclear extracts (20 µg) of A549, H1299, Calu-1, H23 and H441 human lung cancer cells and MCF-7 and MDA-MB-231 (MB-231) breast cancer cells were subjected to immunoblotting for Blimp1 and β-actin, as a control for equal loading. Positions of molecular weight markers are given in the left lane. A representative of two independent experiments with similar results is shown. (B) A549 and (C) H1299 cells were transiently transfected with 10 nM each of siBLIMP1-1 , siBLIMP1-2 or a scrambled negative control siRNA. Upper panels: Forty-eight h after transfection, WCE (30 µg) were subjected to immunoblotting for Blimp1 and β-actin. The bands were quantified using NIH Image J software and Blimp1 expression normalized to β-actin expression. Normalized Blimp1 expression was determined in two independent experiments and the average values are given below the blots. Lower panels: Alternatively, after 24 h, cultures were trypsinized and 1×10 5 cells subjected to a migration assay for 16 h, in triplicate. The average migration from three independent experiments ± SD is presented relative to the negative control siRNA (set at 1.0). P values were calculated using Student's t -test. *, P

    Journal: PLoS ONE

    Article Title: Blimp1 Activation by AP-1 in Human Lung Cancer Cells Promotes a Migratory Phenotype and Is Inhibited by the Lysyl Oxidase Propeptide

    doi: 10.1371/journal.pone.0033287

    Figure Lengend Snippet: Blimp1 is expressed in lung cancer cells and its knockdown reduces migration. (A) Samples of nuclear extracts (20 µg) of A549, H1299, Calu-1, H23 and H441 human lung cancer cells and MCF-7 and MDA-MB-231 (MB-231) breast cancer cells were subjected to immunoblotting for Blimp1 and β-actin, as a control for equal loading. Positions of molecular weight markers are given in the left lane. A representative of two independent experiments with similar results is shown. (B) A549 and (C) H1299 cells were transiently transfected with 10 nM each of siBLIMP1-1 , siBLIMP1-2 or a scrambled negative control siRNA. Upper panels: Forty-eight h after transfection, WCE (30 µg) were subjected to immunoblotting for Blimp1 and β-actin. The bands were quantified using NIH Image J software and Blimp1 expression normalized to β-actin expression. Normalized Blimp1 expression was determined in two independent experiments and the average values are given below the blots. Lower panels: Alternatively, after 24 h, cultures were trypsinized and 1×10 5 cells subjected to a migration assay for 16 h, in triplicate. The average migration from three independent experiments ± SD is presented relative to the negative control siRNA (set at 1.0). P values were calculated using Student's t -test. *, P

    Article Snippet: Antibodies against β-actin (AC-15) and α-tubulin (DM1A) were from Sigma.

    Techniques: Migration, Multiple Displacement Amplification, Molecular Weight, Transfection, Negative Control, Software, Expressing

    Ectopic LOX-PP reduces Blimp1 expression in lung cancer cells. (A) H1299-EV cells, and H1299-LOX-PP4 (PP4) and H1299-LOX-PP7 (PP7) clones, isolated as described previously [25] , were treated in triplicate with 2 µg/ml dox for 48 h. RNA from two independent experiments was subjected to Q-PCR and normalized values for BLIMP1 mRNA relative to GAPDH levels are presented as the mean ± SEM (EV DNA set to 1.0). (B) A549-EV, A549-hLOX-PP, A549-mLOX-PP dox-inducible stable populations were treated with 2 µg/ml dox for 48 h in DMEM supplemented with 0.5% FBS. FBS was added back to 10% and cells incubated overnight. RNA from two independent experiments was subjected to Q-PCR and normalized values for BLIMP1 mRNA relative to GAPDH levels are presented as the mean ± SEM (EV DNA set to 1.0). Samples of medium (5 ml) were subjected to immunoprecipitation followed by immunoblotting using V5 antibody for LOX-PP expression. (C) A549 and H1299 cells were transiently transfected with human LOX-PP cDNA or EV DNA. After 48 h, media and WCE were prepared. Samples of media (50 µl) were subjected to immunoblotting for V5. Samples of WCE (25 µg) were probed for Blimp1 and β-actin, and average normalized Blimp1 values from two independent experiments presented relative to EV DNA, set to 1.0. (D) A549 and H441 cells were treated with purified recombinant LOX-PP protein at a final concentration of 4 or 1 µg/ml, respectively, or the same volume of vehicle (water) in medium with 0.5% FBS. Twenty-four h later, FBS was added back to 10% and cultures incubated overnight. WCE were subjected to immunoblotting for Blimp1, phospho-c-Jun (p-c-Jun), total c-Jun, Fra-1 and Fra-2 and α-tubulin, as a loading control. Normalized Blimp1 and AP-1 subunit values from two independent experiments are presented relative to EV DNA, set to 1.0.

    Journal: PLoS ONE

    Article Title: Blimp1 Activation by AP-1 in Human Lung Cancer Cells Promotes a Migratory Phenotype and Is Inhibited by the Lysyl Oxidase Propeptide

    doi: 10.1371/journal.pone.0033287

    Figure Lengend Snippet: Ectopic LOX-PP reduces Blimp1 expression in lung cancer cells. (A) H1299-EV cells, and H1299-LOX-PP4 (PP4) and H1299-LOX-PP7 (PP7) clones, isolated as described previously [25] , were treated in triplicate with 2 µg/ml dox for 48 h. RNA from two independent experiments was subjected to Q-PCR and normalized values for BLIMP1 mRNA relative to GAPDH levels are presented as the mean ± SEM (EV DNA set to 1.0). (B) A549-EV, A549-hLOX-PP, A549-mLOX-PP dox-inducible stable populations were treated with 2 µg/ml dox for 48 h in DMEM supplemented with 0.5% FBS. FBS was added back to 10% and cells incubated overnight. RNA from two independent experiments was subjected to Q-PCR and normalized values for BLIMP1 mRNA relative to GAPDH levels are presented as the mean ± SEM (EV DNA set to 1.0). Samples of medium (5 ml) were subjected to immunoprecipitation followed by immunoblotting using V5 antibody for LOX-PP expression. (C) A549 and H1299 cells were transiently transfected with human LOX-PP cDNA or EV DNA. After 48 h, media and WCE were prepared. Samples of media (50 µl) were subjected to immunoblotting for V5. Samples of WCE (25 µg) were probed for Blimp1 and β-actin, and average normalized Blimp1 values from two independent experiments presented relative to EV DNA, set to 1.0. (D) A549 and H441 cells were treated with purified recombinant LOX-PP protein at a final concentration of 4 or 1 µg/ml, respectively, or the same volume of vehicle (water) in medium with 0.5% FBS. Twenty-four h later, FBS was added back to 10% and cultures incubated overnight. WCE were subjected to immunoblotting for Blimp1, phospho-c-Jun (p-c-Jun), total c-Jun, Fra-1 and Fra-2 and α-tubulin, as a loading control. Normalized Blimp1 and AP-1 subunit values from two independent experiments are presented relative to EV DNA, set to 1.0.

    Article Snippet: Antibodies against β-actin (AC-15) and α-tubulin (DM1A) were from Sigma.

    Techniques: Expressing, Isolation, Polymerase Chain Reaction, Incubation, Immunoprecipitation, Transfection, Purification, Recombinant, Concentration Assay

    Ectopic AP-1 subunits induce Blimp1 expression. (A) H441 cells, growing in 6-well plates, were transfected with 1 µg of vectors expressing the indicated AP-1 subunits or EV DNA (see bottom) to make a 2 µg total. Upper panel. After 48 h, RNA was isolated and subjected to Q-PCR. The levels of BLIMP1 mRNA normalized to GAPDH mRNA are presented as mean ± SD of three independent experiments. Middle and lower panels. WCE were isolated and subjected to immunoblotting (IB) for Blimp1 (Middle panels), and for c-Jun, Fra-1, Fra-2, c-Fos and β-actin (Lower panels). (L exp., longer exposure; S exp., shorter exposure). Blimp1 levels, normalized to β-actin, were determined as in Fig. 1C and average values from two independent experiments presented relative to EV DNA, set to 1.0. (B) H441 cells were transiently transfected, in triplicate, with 0.3 µg of Blimp1 -Luc, 0.3 µg of MSV-β-gal, and vectors expressing the indicated AP-1 subunits (0.15 µg each) and EV DNA to a total of 1.0 µg DNA. Normalized values of Blimp1 promoter activity are presented as the mean ± SEM from two experiments (EV DNA set to 1.0).

    Journal: PLoS ONE

    Article Title: Blimp1 Activation by AP-1 in Human Lung Cancer Cells Promotes a Migratory Phenotype and Is Inhibited by the Lysyl Oxidase Propeptide

    doi: 10.1371/journal.pone.0033287

    Figure Lengend Snippet: Ectopic AP-1 subunits induce Blimp1 expression. (A) H441 cells, growing in 6-well plates, were transfected with 1 µg of vectors expressing the indicated AP-1 subunits or EV DNA (see bottom) to make a 2 µg total. Upper panel. After 48 h, RNA was isolated and subjected to Q-PCR. The levels of BLIMP1 mRNA normalized to GAPDH mRNA are presented as mean ± SD of three independent experiments. Middle and lower panels. WCE were isolated and subjected to immunoblotting (IB) for Blimp1 (Middle panels), and for c-Jun, Fra-1, Fra-2, c-Fos and β-actin (Lower panels). (L exp., longer exposure; S exp., shorter exposure). Blimp1 levels, normalized to β-actin, were determined as in Fig. 1C and average values from two independent experiments presented relative to EV DNA, set to 1.0. (B) H441 cells were transiently transfected, in triplicate, with 0.3 µg of Blimp1 -Luc, 0.3 µg of MSV-β-gal, and vectors expressing the indicated AP-1 subunits (0.15 µg each) and EV DNA to a total of 1.0 µg DNA. Normalized values of Blimp1 promoter activity are presented as the mean ± SEM from two experiments (EV DNA set to 1.0).

    Article Snippet: Antibodies against β-actin (AC-15) and α-tubulin (DM1A) were from Sigma.

    Techniques: Expressing, Transfection, Isolation, Polymerase Chain Reaction, Activity Assay

    Reduced PAX8 expression leads to decreased expression of BCL2 and WT1. (A) The PAX8 -knockdown (siPAX8) in the A172 glioma cell line by siRNA (PAX8-1) produced a reduction in the BCL2 expression levels. Cells lysates were prepared 36 hours after siRNA transfection, and the PAX8, BCL2, p53, and β-actin (loading control) expression levels were measured by western blot. For controls, A172 cells were transfected with mock-treated (Moc), non-targeting siRNAs (NT1, NT2, and NT3) and scrambled s8-1 siRNA (scPAX8). To ensure the reduction in the glioma cell growth rate associated with the PAX8 -knockdown was not due to p53 function, p53 was also knocked down in A172 cells (sip53) independently or in combination with a PAX8 siRNA (siPAX8 p53). (B ) The PAX8 -knockdown (siPAX8) in the A172 glioma cell line by siRNA (PAX8-1) produced a reduction in the WT1 expression levels. (C) The BCL2 -knockdown produced a similar reduction in the cell growth rate compared to PAX8 -knockdown in the A172 glioma cell line. Cells were transfected with a BCL2 siRNA (siBCL2) or a PAX8 siRNA (PAX8-1, siPAX8). For controls, A172 cells were mock-transfected (Moc) or transfected with non-targeting siRNAs (NT1 and NT3). The percentage of live cells was determined by the trypan blue exclusion assay every 24 hours post-transfection. ( Insert ) Western blotting shows the BCL2-knockdown with a BCL2 siRNA and no BCL2-knockdown in controls; the loading control is β-actin.

    Journal: BMC Cancer

    Article Title: Increased paired box transcription factor 8 has a survival function in Glioma

    doi: 10.1186/1471-2407-14-159

    Figure Lengend Snippet: Reduced PAX8 expression leads to decreased expression of BCL2 and WT1. (A) The PAX8 -knockdown (siPAX8) in the A172 glioma cell line by siRNA (PAX8-1) produced a reduction in the BCL2 expression levels. Cells lysates were prepared 36 hours after siRNA transfection, and the PAX8, BCL2, p53, and β-actin (loading control) expression levels were measured by western blot. For controls, A172 cells were transfected with mock-treated (Moc), non-targeting siRNAs (NT1, NT2, and NT3) and scrambled s8-1 siRNA (scPAX8). To ensure the reduction in the glioma cell growth rate associated with the PAX8 -knockdown was not due to p53 function, p53 was also knocked down in A172 cells (sip53) independently or in combination with a PAX8 siRNA (siPAX8 p53). (B ) The PAX8 -knockdown (siPAX8) in the A172 glioma cell line by siRNA (PAX8-1) produced a reduction in the WT1 expression levels. (C) The BCL2 -knockdown produced a similar reduction in the cell growth rate compared to PAX8 -knockdown in the A172 glioma cell line. Cells were transfected with a BCL2 siRNA (siBCL2) or a PAX8 siRNA (PAX8-1, siPAX8). For controls, A172 cells were mock-transfected (Moc) or transfected with non-targeting siRNAs (NT1 and NT3). The percentage of live cells was determined by the trypan blue exclusion assay every 24 hours post-transfection. ( Insert ) Western blotting shows the BCL2-knockdown with a BCL2 siRNA and no BCL2-knockdown in controls; the loading control is β-actin.

    Article Snippet: Blots were probed with primary antibodies raised against PAX8 (MRQ-50, Cell Marque), Bcl-2 (Clone 124, Dako), p53 (1C12, Cell Signaling Technology, Beverly, MA), WT1 (6FH2, Dako, Glostrup, Denmark) and β-actin (AC-15, Abcam, UK) according to the manufacturers’ instruction, or that optimised in the current study (1:200 dilution for WT1).

    Techniques: Expressing, Produced, Transfection, Western Blot, Trypan Blue Exclusion Assay

    Zey suppresses the Wnt/β-catenin pathway in DU145 cells. (A) DU145 cells were administrated with Zey at 0 (control), 10 (Zey1), 20 (Zey2) and 40 (Zey3) µmol/l for 48 h. Western blot analysis was performed to measure the protein expression of wnt5a, β-catenin and cyclin D1. (B) Quantification of protein was executed using GraphPad Prism software. β-actin was regarded as the internal control. Gray value was assessed and calculated using Quantity One software. ^ P

    Journal: Molecular Medicine Reports

    Article Title: Zeylenone represses the progress of human prostate cancer by downregulating the Wnt/β-catenin pathway

    doi: 10.3892/mmr.2018.9564

    Figure Lengend Snippet: Zey suppresses the Wnt/β-catenin pathway in DU145 cells. (A) DU145 cells were administrated with Zey at 0 (control), 10 (Zey1), 20 (Zey2) and 40 (Zey3) µmol/l for 48 h. Western blot analysis was performed to measure the protein expression of wnt5a, β-catenin and cyclin D1. (B) Quantification of protein was executed using GraphPad Prism software. β-actin was regarded as the internal control. Gray value was assessed and calculated using Quantity One software. ^ P

    Article Snippet: Subsequently, the membrane was blocked with 5% skimmed milk powder at room temperature for 1.5 h. Following blocking, the membrane was incubated with anti-matrix metalloproteinase (MMP)-2 (R & D Systems, Inc.; cat. no. IC903G-100UG; 1:1,000), anti-MMP-9 (Abcam, Cambridge, UK; cat. no. EP1254; 1:500), anti-tissue inhibitor of metalloproteinases-1 (TIMP-1; Abcam; cat. no. ab61224; 1:700), anti-vimentin (R & D Systems, Inc.; cat. no. AF2105; 1:700), anti-epithelial (E)-cadherin (R & D Systems, Inc.; cat. no. MAB1838; 1:1,000), anti-fibronectin 1 (FN1; Abcam; cat. no. ab32419; 1:800), anti-collagen-1 (Abcam; cat. no. ab90395; 1:600), anti-wnt5a (Abcam; cat. no. ab174963; 1:1,000), anti-β-catenin (R & D Systems, Inc.; cat. no. AF1329; 1:1,000), anti-cyclin D1 (R & D Systems, Inc.; cat. no. MAB4314; 1:900) and anti-β-actin (Abcam; cat. no. ab13772; 1:800) on the rocking table at 4°C for 24 h. The membrane was subsequently incubated in corresponding horseradish peroxidase-conjugated secondary antibodies [rabbit anti-mouse immunoglobulin (Ig)G; Cell Signaling Technology, Inc., Danvers, MA, USA; cat. no. 58802; 1:8,000; mouse anti-rabbit IgG; Cell Signaling Technology, Inc.; cat. no. 5127; 1:7,000; goat anti-mouse IgG; Abcam; cat. no. ab6785; 1:7,000] at 37°C for 1 h. The protein was visualized using an enhanced chemiluminescence detection kit (Beyotime Institute of Biotechnology). β-actin was used as an internal control.

    Techniques: Western Blot, Expressing, Software

    Zey regulates extracellular matrix-associated factors in DU145 cells. (A) DU145 cells exposed to Zey at 0 (control), 10 (Zey1), 20 (Zey2) and 40 (Zey3) µmol/l for 48 h. Expression levels of MMP-2 and MMP-9 were detected using an ELISA. (B) mRNA expressions of MMP-2, MMP-9, TIMP-1, FN-1 and collagen-1 were measured by a reverse transcription-quantitative polymerase chain reaction assay. (C) Protein expression of MMP-2, MMP-9, TIMP-1, FN-1 and collagen-1 were measured by western blot analysis and normalized to β-actin expression. Gray value was evaluated and quantified using Quantity One software. ^ P

    Journal: Molecular Medicine Reports

    Article Title: Zeylenone represses the progress of human prostate cancer by downregulating the Wnt/β-catenin pathway

    doi: 10.3892/mmr.2018.9564

    Figure Lengend Snippet: Zey regulates extracellular matrix-associated factors in DU145 cells. (A) DU145 cells exposed to Zey at 0 (control), 10 (Zey1), 20 (Zey2) and 40 (Zey3) µmol/l for 48 h. Expression levels of MMP-2 and MMP-9 were detected using an ELISA. (B) mRNA expressions of MMP-2, MMP-9, TIMP-1, FN-1 and collagen-1 were measured by a reverse transcription-quantitative polymerase chain reaction assay. (C) Protein expression of MMP-2, MMP-9, TIMP-1, FN-1 and collagen-1 were measured by western blot analysis and normalized to β-actin expression. Gray value was evaluated and quantified using Quantity One software. ^ P

    Article Snippet: Subsequently, the membrane was blocked with 5% skimmed milk powder at room temperature for 1.5 h. Following blocking, the membrane was incubated with anti-matrix metalloproteinase (MMP)-2 (R & D Systems, Inc.; cat. no. IC903G-100UG; 1:1,000), anti-MMP-9 (Abcam, Cambridge, UK; cat. no. EP1254; 1:500), anti-tissue inhibitor of metalloproteinases-1 (TIMP-1; Abcam; cat. no. ab61224; 1:700), anti-vimentin (R & D Systems, Inc.; cat. no. AF2105; 1:700), anti-epithelial (E)-cadherin (R & D Systems, Inc.; cat. no. MAB1838; 1:1,000), anti-fibronectin 1 (FN1; Abcam; cat. no. ab32419; 1:800), anti-collagen-1 (Abcam; cat. no. ab90395; 1:600), anti-wnt5a (Abcam; cat. no. ab174963; 1:1,000), anti-β-catenin (R & D Systems, Inc.; cat. no. AF1329; 1:1,000), anti-cyclin D1 (R & D Systems, Inc.; cat. no. MAB4314; 1:900) and anti-β-actin (Abcam; cat. no. ab13772; 1:800) on the rocking table at 4°C for 24 h. The membrane was subsequently incubated in corresponding horseradish peroxidase-conjugated secondary antibodies [rabbit anti-mouse immunoglobulin (Ig)G; Cell Signaling Technology, Inc., Danvers, MA, USA; cat. no. 58802; 1:8,000; mouse anti-rabbit IgG; Cell Signaling Technology, Inc.; cat. no. 5127; 1:7,000; goat anti-mouse IgG; Abcam; cat. no. ab6785; 1:7,000] at 37°C for 1 h. The protein was visualized using an enhanced chemiluminescence detection kit (Beyotime Institute of Biotechnology). β-actin was used as an internal control.

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

    Zey regulates epithelial-mesenchymal transition-associated factors in DU145 cells. (A) DU145 cells were administered with Zey at 0 (control), 10 (Zey1), 20 (Zey2) and 40 (Zey3) µmol/l for 48 h. Reverse transcription-quantitative polymerase chain reaction was conducted to assess the mRNA expression of vimentin and E-cadherin. (B) Western blot analysis was used to determine the protein expression of vimentin and E-cadherin. β-actin was presented as the internal control. Gray value was assessed and calculated using Quantity One software. ^ P

    Journal: Molecular Medicine Reports

    Article Title: Zeylenone represses the progress of human prostate cancer by downregulating the Wnt/β-catenin pathway

    doi: 10.3892/mmr.2018.9564

    Figure Lengend Snippet: Zey regulates epithelial-mesenchymal transition-associated factors in DU145 cells. (A) DU145 cells were administered with Zey at 0 (control), 10 (Zey1), 20 (Zey2) and 40 (Zey3) µmol/l for 48 h. Reverse transcription-quantitative polymerase chain reaction was conducted to assess the mRNA expression of vimentin and E-cadherin. (B) Western blot analysis was used to determine the protein expression of vimentin and E-cadherin. β-actin was presented as the internal control. Gray value was assessed and calculated using Quantity One software. ^ P

    Article Snippet: Subsequently, the membrane was blocked with 5% skimmed milk powder at room temperature for 1.5 h. Following blocking, the membrane was incubated with anti-matrix metalloproteinase (MMP)-2 (R & D Systems, Inc.; cat. no. IC903G-100UG; 1:1,000), anti-MMP-9 (Abcam, Cambridge, UK; cat. no. EP1254; 1:500), anti-tissue inhibitor of metalloproteinases-1 (TIMP-1; Abcam; cat. no. ab61224; 1:700), anti-vimentin (R & D Systems, Inc.; cat. no. AF2105; 1:700), anti-epithelial (E)-cadherin (R & D Systems, Inc.; cat. no. MAB1838; 1:1,000), anti-fibronectin 1 (FN1; Abcam; cat. no. ab32419; 1:800), anti-collagen-1 (Abcam; cat. no. ab90395; 1:600), anti-wnt5a (Abcam; cat. no. ab174963; 1:1,000), anti-β-catenin (R & D Systems, Inc.; cat. no. AF1329; 1:1,000), anti-cyclin D1 (R & D Systems, Inc.; cat. no. MAB4314; 1:900) and anti-β-actin (Abcam; cat. no. ab13772; 1:800) on the rocking table at 4°C for 24 h. The membrane was subsequently incubated in corresponding horseradish peroxidase-conjugated secondary antibodies [rabbit anti-mouse immunoglobulin (Ig)G; Cell Signaling Technology, Inc., Danvers, MA, USA; cat. no. 58802; 1:8,000; mouse anti-rabbit IgG; Cell Signaling Technology, Inc.; cat. no. 5127; 1:7,000; goat anti-mouse IgG; Abcam; cat. no. ab6785; 1:7,000] at 37°C for 1 h. The protein was visualized using an enhanced chemiluminescence detection kit (Beyotime Institute of Biotechnology). β-actin was used as an internal control.

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Software

    Immunocytochemical characterization. Confluent HUVEC cell layers after 24 h of NAC supplemented culture. In 15 mM NAC enriched cultivation, endothelial cells changed their morphology from typical cobblestone pattern (a–d) to an elongated, quasi-fusiform configuration (i–l). A slight cellular rearrangement was already detectable at 10 mM (e–h). Cells were positive for common endothelial cell markers CD31 (PECAM-1) and Von-Willebrand-Factor (vWF). Nuclei were counterstained with DAPI. All cells were negative for alpha smooth muscle actin (αSMA), a common myofibroblast marker (scale bar: 100 µ m).

    Journal: Cellular and Molecular Bioengineering

    Article Title: Towards a Biohybrid Lung Assist Device: N-Acetylcysteine Reduces Oxygen Toxicity and Changes Endothelial Cells’ Morphology

    doi: 10.1007/s12195-016-0473-4

    Figure Lengend Snippet: Immunocytochemical characterization. Confluent HUVEC cell layers after 24 h of NAC supplemented culture. In 15 mM NAC enriched cultivation, endothelial cells changed their morphology from typical cobblestone pattern (a–d) to an elongated, quasi-fusiform configuration (i–l). A slight cellular rearrangement was already detectable at 10 mM (e–h). Cells were positive for common endothelial cell markers CD31 (PECAM-1) and Von-Willebrand-Factor (vWF). Nuclei were counterstained with DAPI. All cells were negative for alpha smooth muscle actin (αSMA), a common myofibroblast marker (scale bar: 100 µ m).

    Article Snippet: After washing cells, now using permeabilizing washing buffer (0.1% Triton in PBS), staining procedure for intracellular markers was undertaken using primary antibody rabbit-against vWF (Dako, A0082, 1:100) with secondary antibody goat-anti-rabbit (Alexa Fluor 594, Invitrogen A11012, 1:400) and primary antibody mouse against αSMA (Sigma, A2547) with secondary antibody goat-anti-mouse (Invitrogen, Alexa Fluor 594, A11005).

    Techniques: Marker

    CD47 is highly expressed in osteosarcoma Quantitative RT-PCR A. and immunoblot analysis B. showed CD47 expression in ten freshly isolated osteosarcoma tissues and their adjacent nontumorous tissues. Beta-actin was treated as the reference control. CD47 immunostaining C. showed higher of CD47 expression in osteosarcoma of the same samples with or without metastasis and their adjacent nontumorous tissues. The tissues were counterstained with DAPI Fluorescent Stain. Representative images are shown here. (magnification × 100). D. Flow-cytometry analysis of CD47 and CD44 expressions on osteosarcoma tumor cells and quantification of CD44 cells also expressing CD47.

    Journal: Oncotarget

    Article Title: CD47 blockade inhibits tumor progression human osteosarcoma in xenograft models

    doi:

    Figure Lengend Snippet: CD47 is highly expressed in osteosarcoma Quantitative RT-PCR A. and immunoblot analysis B. showed CD47 expression in ten freshly isolated osteosarcoma tissues and their adjacent nontumorous tissues. Beta-actin was treated as the reference control. CD47 immunostaining C. showed higher of CD47 expression in osteosarcoma of the same samples with or without metastasis and their adjacent nontumorous tissues. The tissues were counterstained with DAPI Fluorescent Stain. Representative images are shown here. (magnification × 100). D. Flow-cytometry analysis of CD47 and CD44 expressions on osteosarcoma tumor cells and quantification of CD44 cells also expressing CD47.

    Article Snippet: After stripping with Restore Western Blot Stripping Buffer (Pierce) for 20 min at room temperature, membranes were processed similarly with anti-beta-actin antibody (1:2, 000 dilution, Abcam) as a loading control.

    Techniques: Quantitative RT-PCR, Expressing, Isolation, Immunostaining, Staining, Flow Cytometry, Cytometry