Journal: The Journal of Biological Chemistry

Article Title: Novel Ubiquitin-derived High Affinity Binding Proteins with Tumor Targeting Properties *

doi: 10.1074/jbc.M113.519884

Figure Lengend Snippet: Target binding specificity of 77405 as determined by Western blot analyses. A , SDS-PAGE of analyzed samples. B , detection of ED-B target using the 77405 variant, a rabbit anti-Strep-tag, and an anti-rabbit POD-conjugated antibody. C , detection of fibronectin using a mouse anti-human fibronectin and an anti-mouse POD-conjugated antibody. D , control blot using the secondary anti-rabbit antibody yielding a single band at 40 kDa due to nonspecific binding of the secondary antibody. E , detection of β-tubulin using an anti-tubulin antibody. For all figures, lanes were loaded with the following samples: 5 μg of cellular fibronectin ( lane 1 ), 2 μg of cellular fibronectin ( lane 2 ), 0.5 μg of cellular fibronectin ( lane 3 ), 50 ng of 67B89 ( lane 4 ), 50 ng of 6789 ( lane 5 ), 60 μg of protein from HT29 cell lysate ( lane 6 ), 60 μg of protein from NHDF cell lysate ( lane 7 ), and 60 μg of protein from Wi38 cell lysate ( lane 8 ).

Article Snippet: Cell pellets of Wi38 (CCL-75, LGC Standards GmbH, Wesel, Germany), NHDF (C12302, Promocell, Heidelberg, Germany), and HT29 (ACC-299, DSMZ, Heidelberg, Germany) were resuspended in cell extraction buffer (Invitrogen), and cell lysates were prepared according to the suppliers' instructions for Western blot analysis.

Techniques: Binding Assay, Western Blot, SDS Page, Variant Assay, Strep-tag, Control

Journal: Frontiers in Oncology

Article Title: Origanum majorana Ethanolic Extract Promotes Colorectal Cancer Cell Death by Triggering Abortive Autophagy and Activation of the Extrinsic Apoptotic Pathway

doi: 10.3389/fonc.2019.00795

Figure Lengend Snippet: Origanum majorana ethanolic extract inhibits cellular viability of colorectal cancer cells. (A) Exponentially growing HT-29 and (B) Caco-2 colon cancer cells were treated with and without of various concentration (0, 150, 300, 450, and 600 μg/mL) OME for 24 and 48 h. Viability was measured using a colorimetric assay as described in section Materials and Methods. Values are represented as mean ± SD of n = 4 (* p < 0.05 and *** p < 0.001). (C) HT-29 cells were exposed to OME for 24 and 48 h and number of viable cells, using a fluorescent dye, was monitored as described in section Materials and Methods using the Muse Cell Analyzer (Millipore). Data represent the mean ± SD of n = 3 carried out in triplicate.

Article Snippet: Human colon cancer cells HT-29 (Cat# 300215) and CaCo-2 (Cat # 300137) were purchased from CLS (cell lines service, Germany).

Techniques: Concentration Assay, Colorimetric Assay

Journal: Frontiers in Oncology

Article Title: Origanum majorana Ethanolic Extract Promotes Colorectal Cancer Cell Death by Triggering Abortive Autophagy and Activation of the Extrinsic Apoptotic Pathway

doi: 10.3389/fonc.2019.00795

Figure Lengend Snippet: Origanum majorana inhibits HT-29 colony growth. (A–C) Inhibition of formed HT-29 colony growth by various concentrations of OME (0, 150, 300, 450, and 600 μg/mL) was assessed by measuring the number and average size (surface area) of the colonies obtained in control and OME-treated plate as described in section Materials and methods. Values are represented as mean ± SD of n = 3 (* p < 0.05 and ** p < 0.005).

Article Snippet: Human colon cancer cells HT-29 (Cat# 300215) and CaCo-2 (Cat # 300137) were purchased from CLS (cell lines service, Germany).

Techniques: Inhibition

Journal: Frontiers in Oncology

Article Title: Origanum majorana Ethanolic Extract Promotes Colorectal Cancer Cell Death by Triggering Abortive Autophagy and Activation of the Extrinsic Apoptotic Pathway

doi: 10.3389/fonc.2019.00795

Figure Lengend Snippet: OME induces a mitotic arrest in HT-29 cells. (A,B) Cell cycle distribution analysis in HT-29 cells treated with and without OME (0, 150, 300, 450, and 600 μg/mL) for 24 h. Values are represented as mean ± SD of n = 3 (* p < 0.05, ** p < 0.005, and *** p < 0.001). (C) Alteration in proteins associated with cell cycle regulation in OME-treated HT-29 cells.

Article Snippet: Human colon cancer cells HT-29 (Cat# 300215) and CaCo-2 (Cat # 300137) were purchased from CLS (cell lines service, Germany).

Techniques:

Journal: Frontiers in Oncology

Article Title: Origanum majorana Ethanolic Extract Promotes Colorectal Cancer Cell Death by Triggering Abortive Autophagy and Activation of the Extrinsic Apoptotic Pathway

doi: 10.3389/fonc.2019.00795

Figure Lengend Snippet: Activation of extrinsic apoptotic pathway and upregulation of TNF-α in OME-treated HT-29 cells. (A) Western blot analysis of caspase 3, 7, and 8 activation and PARP cleavage in HT-29 cells. Cells were treated with or without increasing concentration (0, 150, 300, 450, and 600 μg/mL) of OME for 48 h, then whole cell proteins were extracted and subjected to Western blot analysis for the markers of apoptosis (B) Western blot analysis of TNF-α (C) Western blot analysis of cleaved PARP in cells pretreated for 1 h with and without Z-VAD-FMK (50 μM) followed by treatment with OME (450 μg/mL) for 48 h. (D) Inhibition of apoptosis has a minimal effect of OME-induced cell death. HT-29 cells were pretreated with Z-VAD-FMK as described above and then treated for 48 h with 450 μg/mL OME. Cell viability was determined as described in section Material and Methods. Values are represented as mean ± SD of n = 3 (* p < 0.05 and *** p < 0.001).

Article Snippet: Human colon cancer cells HT-29 (Cat# 300215) and CaCo-2 (Cat # 300137) were purchased from CLS (cell lines service, Germany).

Techniques: Activation Assay, Western Blot, Concentration Assay, Inhibition

Journal: Frontiers in Oncology

Article Title: Origanum majorana Ethanolic Extract Promotes Colorectal Cancer Cell Death by Triggering Abortive Autophagy and Activation of the Extrinsic Apoptotic Pathway

doi: 10.3389/fonc.2019.00795

Figure Lengend Snippet: OME induces abortive autophagy in HT-29 cells. Western blotting analysis of LC3II, p62(SQSTM1), and Beclin-1 expression OME-treated HT-29 cells. Cells were treated with or without increasing concentration (0, 150, 300, 450, and 600 μg/mL) of OME for 48 h, then whole cell proteins were extracted and subjected to Western blot analysis, as described in section Materials and Methods, for LC3II, 62(SQSTM1), and Beclin-1.

Article Snippet: Human colon cancer cells HT-29 (Cat# 300215) and CaCo-2 (Cat # 300137) were purchased from CLS (cell lines service, Germany).

Techniques: Western Blot, Expressing, Concentration Assay

Journal: Frontiers in Oncology

Article Title: Origanum majorana Ethanolic Extract Promotes Colorectal Cancer Cell Death by Triggering Abortive Autophagy and Activation of the Extrinsic Apoptotic Pathway

doi: 10.3389/fonc.2019.00795

Figure Lengend Snippet: OME induces DNA damage in response to OME treatment in HT-29 cells. HT-29 cells were treated with increasing concentrations (0, 150, 300, 450, and 600 μg/mL) of OME for 48 h. DNA damage was examined by western blotting by measuring the level of phosphorylated H2AX.

Article Snippet: Human colon cancer cells HT-29 (Cat# 300215) and CaCo-2 (Cat # 300137) were purchased from CLS (cell lines service, Germany).

Techniques: Western Blot

Journal: Frontiers in Oncology

Article Title: Origanum majorana Ethanolic Extract Promotes Colorectal Cancer Cell Death by Triggering Abortive Autophagy and Activation of the Extrinsic Apoptotic Pathway

doi: 10.3389/fonc.2019.00795

Figure Lengend Snippet: DNA damage and autophagy precedes apoptosis in OME-treated HT-29 cells. (A) Time-course analysis, by Western blotting, of PARP and caspase 8 cleavage, LC3-II, p62 (SQSTM1), γH2AX, and H3pser10 accumulation in OME-treated HT-29 cells. Cells were treated with 450 μg/mL OME and proteins were extracted at the indicated time-points (0, 4, 8, 24, and 48 h) as described in section Materials and Methods. (B) Western blot analysis of γH2AX accumulation in HT-29 cells pre-treated with 3MA. Cells were pretreated with or without 3-MA (5 mM) for 1 h and then OME (450 μg/mL) was added, and cells were incubated for 48 h.

Article Snippet: Human colon cancer cells HT-29 (Cat# 300215) and CaCo-2 (Cat # 300137) were purchased from CLS (cell lines service, Germany).

Techniques: Western Blot, Incubation

Journal: Frontiers in Oncology

Article Title: Origanum majorana Ethanolic Extract Promotes Colorectal Cancer Cell Death by Triggering Abortive Autophagy and Activation of the Extrinsic Apoptotic Pathway

doi: 10.3389/fonc.2019.00795

Figure Lengend Snippet: Inhibition of autophagy decreases OME-induced cell death in HT-29 cells. (A) Analysis of LC3-II and cleaved PARP accumulation in HT-29 cells pre-treated with 3-MA. Cells were pretreated with or without 3-MA (5 mM) for 1 h and then OME (450 μg/mL) was added, and cells were incubated for 48 h. (B) Inhibition of autophagy reduces cell death induced by OME. HT-29 cells were pretreated with 3-MA for 1 h and then for 48 h with 450 μg/mL OME. Cell viability was determined as described in Material and Methods. Values are represented as mean ± SD of n = 3 (*** p < 0.001).

Article Snippet: Human colon cancer cells HT-29 (Cat# 300215) and CaCo-2 (Cat # 300137) were purchased from CLS (cell lines service, Germany).

Techniques: Inhibition, Incubation

Journal: Frontiers in Oncology

Article Title: Origanum majorana Ethanolic Extract Promotes Colorectal Cancer Cell Death by Triggering Abortive Autophagy and Activation of the Extrinsic Apoptotic Pathway

doi: 10.3389/fonc.2019.00795

Figure Lengend Snippet: Downregulation of survivin by OME in HT-29 cells. HT-29 cells were treated with increasing concentrations (0, 150, 300, 450, and 600 μg/mL) of OME for 48 h and the level of survivin was assessed by Western blotting.

Article Snippet: Human colon cancer cells HT-29 (Cat# 300215) and CaCo-2 (Cat # 300137) were purchased from CLS (cell lines service, Germany).

Techniques: Western Blot

Journal: Viruses

Article Title: Establishing a New Platform to Investigate the Efficacy of Oncolytic Virotherapy in a Human Ex Vivo Peritoneal Carcinomatosis Model

doi: 10.3390/v15020363

Figure Lengend Snippet: Virotherapeutic treatment of GFP/luc-labeled human HT-29 tumor cells in cell culture with GLV-0b347. ( A ) Schematic illustration of the three-step virotherapeutic process and associated detection capabilities: (1) GFP/luc-labeled HT-29 tumor cells were seeded into a 24-well cell culture plate. Successful plating of the cells can be verified by the determination of GFP fluorescence. (2) The treatment of HT-29 cells with oncolytic viruses encoding a red-fluorescent marker protein. The successful infection of the tumor cells can be verified by the determination of red fluorescence. (3)/(4) The viral oncolysis can be determined by a decrease in GFP as well as in luciferase activity. Over time, enhanced red fluorescence indicates an increasing number of tumor cells being infected by the red-fluorescence marker gene encoding virotherapeutic compounds. ( B ) The fluorescence images of HT-29 GFP/luc-labeled cells at 72 h postinfection (hpi) with GLV-0b347 at different multiplicities of infection (MOIs), as depicted. BF, brightfield; OL, overlay of GFP and TurboFP635 signal.

Article Snippet: The human GFP/Luciferase (luc) dual-labeled HT-29 cancer cell line was purchased from GeneCopoeia TM (Cat. SCL-C06-HLG, Rockville, MD, USA) and authenticated by short tandem repeat analysis (Eurofins, Ebersberg, Germany).

Techniques: Labeling, Cell Culture, Fluorescence, Marker, Infection, Luciferase, Activity Assay

Journal: Viruses

Article Title: Establishing a New Platform to Investigate the Efficacy of Oncolytic Virotherapy in a Human Ex Vivo Peritoneal Carcinomatosis Model

doi: 10.3390/v15020363

Figure Lengend Snippet: Virotherapeutic treatment of GFP/luc-labeled human HT-29 tumor cells in cell culture with MeV-DsRed. Fluorescence images of HT-29 GFP/luc-labeled cells at 72 h postinfection (hpi) with MeV-DsRed at different multiplicities of infection (MOIs), as depicted. BF, brightfield; OL, overlay of GFP and DsRed signal.

Article Snippet: The human GFP/Luciferase (luc) dual-labeled HT-29 cancer cell line was purchased from GeneCopoeia TM (Cat. SCL-C06-HLG, Rockville, MD, USA) and authenticated by short tandem repeat analysis (Eurofins, Ebersberg, Germany).

Techniques: Labeling, Cell Culture, Fluorescence, Infection

Journal: Viruses

Article Title: Establishing a New Platform to Investigate the Efficacy of Oncolytic Virotherapy in a Human Ex Vivo Peritoneal Carcinomatosis Model

doi: 10.3390/v15020363

Figure Lengend Snippet: Comparison of different detection options for the oncolytic activity of virotherapeutic compounds GLV-0b347 ( A , images to the left) and MeV-DsRed ( B , images to the right) in HT-29 GFP/luc tumor cells. HT-29 GFP/luc cells were infected with GLV-0b347 ( A ) or MeV-DsRed ( B ) at different multiplicities of infection (MOIs) ranging from 0.0001 to 1 for GLV-0b347, from 0.001 to 10 for MeV-DsRed, or remained uninfected (MOCK). At 72 h postinfection (hpi), remaining tumor cell masses were determined by either (i) SRB viability assays, (ii) the measurement of the luciferase activity, or (iii) the quantification of the GFP or red-fluorescence intensity. Each measurement was calculated relative to the MOCK control. The mean ± SD of at least two independent experiments performed in triplicate is shown. ANOVA test relative to MOCK-infected control: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: The human GFP/Luciferase (luc) dual-labeled HT-29 cancer cell line was purchased from GeneCopoeia TM (Cat. SCL-C06-HLG, Rockville, MD, USA) and authenticated by short tandem repeat analysis (Eurofins, Ebersberg, Germany).

Techniques: Activity Assay, Infection, Luciferase, Fluorescence

Journal: Viruses

Article Title: Establishing a New Platform to Investigate the Efficacy of Oncolytic Virotherapy in a Human Ex Vivo Peritoneal Carcinomatosis Model

doi: 10.3390/v15020363

Figure Lengend Snippet: Human ex vivo peritoneum model and schematic illustration of the three-step virotherapeutic process in co-cultures with GFP/luc-labeled human HT-29 tumor cells. ( A ) Photographic image of the human ex vivo peritoneal model cultivated between stainless steel rings in a 24-well plate. ( B ) Photographic image of the peritoneum in the ex vivo model through a light microscope. ( C ) (1) Preparation of co-cultures of the peritoneum from noncancer patients and human GFP/luc-labeled HT-29 tumor cells. Successful plating of the cells can be verified by fluorescence microscopy. (2) Virotherapeutic treatment of co-cultures with oncolytic viruses carrying a red-fluorescent marker protein. Successful infection of the tumor cells can be verified by the determination of red fluorescence via fluorescence microscopy. (3) Viral oncolysis can be determined by a decrease in GFP and red fluorescence as well as by a decrease in luciferase activity.

Article Snippet: The human GFP/Luciferase (luc) dual-labeled HT-29 cancer cell line was purchased from GeneCopoeia TM (Cat. SCL-C06-HLG, Rockville, MD, USA) and authenticated by short tandem repeat analysis (Eurofins, Ebersberg, Germany).

Techniques: Ex Vivo, Labeling, Light Microscopy, Fluorescence, Microscopy, Marker, Infection, Luciferase, Activity Assay

Journal: Viruses

Article Title: Establishing a New Platform to Investigate the Efficacy of Oncolytic Virotherapy in a Human Ex Vivo Peritoneal Carcinomatosis Model

doi: 10.3390/v15020363

Figure Lengend Snippet: Virotherapeutic treatment of PC models with recombinant vaccinia virus GLV-0b347. The fluorescence images of GLV-0b347-infected co-cultures (( A ); 1.5 × 10 6 plaque-forming units (PFU)) or MOCK-infected ( B ) co-cultures consisting of the peritoneum of noncancer patients and adherently growing GFP/luc-labeled human HT-29 tumor cells at days 2, 4, and 7 postinfection (dpi); original magnification 4×. ( C ) Luciferase activity of GFP/luc–HT-29 cells growing on peritoneum at 2, 4, and 7 dpi, either GLV-0b347-infected or MOCK-infected. Each measurement is normalized to the MOCK control. The mean ± SD of one experiment performed in triplicates is shown. t -test relative to MOCK-infected control: * p < 0.05 and ** p < 0.01. ns; not significant. ( D ) The hematoxylin and eosin staining of human peritoneal tissue with and w/o co-culture of GFP/luc-labeled HT-29 cells at 7 dpi, either GLV-0b347-infected or MOCK-infected. ( E ) The EpCAM staining of peritoneal tissue with the co-culture of HT-29 cells at 7 dpi, either GLV-0b347-infected or MOCK-infected. ( F ) The vaccinia virus staining of peritoneal tissue with co-culture of HT-29 cells at 7 dpi, either GLV-0b347-infected or MOCK-infected. Experiments were conducted with the peritoneal tissue from different patients and show representative data from at least three different experiments. P; peritoneum. Black arrows indicate intact or infected and lysed HT-29 cells on the surface of the peritoneum. Scale bars represent 100 μm.

Article Snippet: The human GFP/Luciferase (luc) dual-labeled HT-29 cancer cell line was purchased from GeneCopoeia TM (Cat. SCL-C06-HLG, Rockville, MD, USA) and authenticated by short tandem repeat analysis (Eurofins, Ebersberg, Germany).

Techniques: Recombinant, Fluorescence, Infection, Labeling, Luciferase, Activity Assay, Staining, Co-Culture Assay

Journal: Viruses

Article Title: Establishing a New Platform to Investigate the Efficacy of Oncolytic Virotherapy in a Human Ex Vivo Peritoneal Carcinomatosis Model

doi: 10.3390/v15020363

Figure Lengend Snippet: Virotherapeutic treatment of PC models with recombinant measles vaccine virus MeV-DsRed. The fluorescence images of MeV-DsRed-infected co-cultures (( A ); 1.5 × 10 6 plaque-forming units (PFU)) or MOCK-infected ( B ) co-cultures consisting of the peritoneum of noncancer patients and GFP/luc-labeled human HT-29 tumor cells at days 2, 4, and 7 postinfection (dpi); original magnification 4×. ( C ) The luciferase activity of GFP/luc–HT-29 cells growing on peritoneum at 2, 4, and 7 dpi, either MeV-DsRed-infected or MOCK-infected. Each measurement is normalized to the MOCK control. The mean ± SD of one experiment performed in triplicates is shown. t -test relative to MOCK-infected control: * p < 0.05. ns; not significant. ( D ) The hematoxylin and eosin staining of human peritoneal tissue with and w/o co-culture of GFP/luc-labeled HT-29 tumor cells at 7 dpi with MeV-DsRed or MOCK infection. ( E ) The EpCAM staining of peritoneal tissue with co-culture of HT-29 cells at 7 dpi with MeV-DsRed or MOCK infection. ( F ) The MeV staining of peritoneal tissue with co-culture of HT-29 cells at 7 dpi with MeV-DsRed or MOCK infection. The experiments were conducted with peritoneal tissue from different patients and show representative data from at least three different experiments. P; peritoneum. Black arrows indicate intact or infected and lysed HT-29 tumor cells on the surface of the peritoneum. Scale bars represent 100 μm.

Article Snippet: The human GFP/Luciferase (luc) dual-labeled HT-29 cancer cell line was purchased from GeneCopoeia TM (Cat. SCL-C06-HLG, Rockville, MD, USA) and authenticated by short tandem repeat analysis (Eurofins, Ebersberg, Germany).

Techniques: Recombinant, Fluorescence, Infection, Labeling, Luciferase, Activity Assay, Staining, Co-Culture Assay