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: Autophagy

Article Title: ALDH1A1 and HLTF modulate the activity of lysosomal autophagy inhibitors in cancer cells

doi: 10.1080/15548627.2017.1377377

Figure Lengend Snippet: Expression levels of ALDH1A1 and HLTF predict sensitivity to HCQ in cancer cell lines. (A) MTT (72 h) in colon and lung cancer cells. (B) Differentially expressed genes in HCQ-S (HT29) and HCQ-R (HCT15) colon cancer cells. (C) HCQ IC50 and Hill Slope for 33 human cancer cell lines. Blue dots indicate sensitive cell lines (<16 µM IC50); green indicates intermediate resistant cell lines (IC50 > 16 µM, Slope > −2.1); red indicates resistant cell lines (IC50 > 16, slope < − 2.1) (D) Protein expression detected by western blot of the 2 most upregulated (ALDH1A1, LYZ) and the 2 most downregulated (ABCB1, HLTF) genes in HCQ-sensitive (Sen), HCQ-intermediate resistant (Int Res), and HCQ-resistant (Res) cells. ANOVA indicates no single gene predicts sensitivity or resistance. (E) CART analysis of expression level of 4 genes (ALDH1A1, LYZ, HLTF, ABCB1) identifies a 2-gene signature that is sufficient to predict all sensitive cell lines. Exp.: expression as detected by fluorescence intensity of the band/ control.

Article Snippet: For detection of RAD52 foci, HT29 cells were transfected with pCMV6 empty vector (Origene, PS100001) and pCMV6 HLTF (Origene, RC207920) and then the indicated cells were grown on chamber slides overnight to reach 50% confluence.

Techniques: Expressing, Western Blot, Fluorescence

Journal: Autophagy

Article Title: ALDH1A1 and HLTF modulate the activity of lysosomal autophagy inhibitors in cancer cells

doi: 10.1080/15548627.2017.1377377

Figure Lengend Snippet: ALDH1A1 levels control entry and activity of chloroquine derivatives in cancer cells. (A-B) Aldeflour assay shows (A) CQ derivatives produce no impairment of ALDH1 enzyme function, (B) siRNA against ALDH1A1 impairs enzymatic function. (C) DC341-C3 strucutre. (D) Fluorescence microscopy of DC340-Cy3 (red fluorescence) in A375 cells treated with nontargeting siRNA (NT) or siALDH1A1; or vehicle and DEAB for 24 h. Mean +/− SD from multiple experiments. (E) DC340-Cy3 fluorescence following SiNT or SiALDH1A1 in HT29 in the presence or absence of verapamil. (F) CD340-Cy3 in A375P cells transiently transfected with control or ALDH1A1-expressing vector. (G) Immunoblotting against autophagy markers in HCT15 cells transfected with control or ADLH1A1-expressing vector +- HCQ. Mean +/− SD for band quantification from multiple experiments shown. (H) LysoSensor fluorescence (green) of HCT15 and HT29 cells transfected with control or ALDH1A1-expressing vector, or siNT or siALDHA1, respectively. (I-K) 72-h MTT mean +/− SD. (I) Knockdown of ALDH1A1 promotes resistance to HCQ. (J) DEAB co-treatment promotes resistance to HCQ. (K) Overexpression of ALDH1A1 promotes HCQ-sensitivity. *p < 0.05.

Article Snippet: For detection of RAD52 foci, HT29 cells were transfected with pCMV6 empty vector (Origene, PS100001) and pCMV6 HLTF (Origene, RC207920) and then the indicated cells were grown on chamber slides overnight to reach 50% confluence.

Techniques: Activity Assay, Fluorescence, Microscopy, Transfection, Expressing, Plasmid Preparation, Western Blot, Over Expression

Journal: Autophagy

Article Title: ALDH1A1 and HLTF modulate the activity of lysosomal autophagy inhibitors in cancer cells

doi: 10.1080/15548627.2017.1377377

Figure Lengend Snippet: Forced expression or knockdown of HLTF reverses HCQ sensitivity or resistance in cancer cells. (A) 2-wk colony formation assay, mean +/− SD. (B-C) immunoblotting and MTT assay of stable transfectants. (D) HCQ treatment +/- 5-azacytadine (aza). (E) Effects of knockdown of HLTF using shRNA on HCQ-R HCT15 cells. (F) Effects of overexpression or knockdown of HLTF on DC340-Cy3 uptake. (G) Sensitivity of HCQ and other lysosomal autophagy inhibitors depends on HLTF expression. HT29 vector (blue) and HT29HLTF (red) cells were treated as indicated. MTT at 72 h was measured. *p < 0.05.

Article Snippet: For detection of RAD52 foci, HT29 cells were transfected with pCMV6 empty vector (Origene, PS100001) and pCMV6 HLTF (Origene, RC207920) and then the indicated cells were grown on chamber slides overnight to reach 50% confluence.

Techniques: Expressing, Colony Assay, Western Blot, MTT Assay, shRNA, Over Expression, Plasmid Preparation

Journal: Autophagy

Article Title: ALDH1A1 and HLTF modulate the activity of lysosomal autophagy inhibitors in cancer cells

doi: 10.1080/15548627.2017.1377377

Figure Lengend Snippet: Overexpression of HLTF promotes resistance to Lys05 in HCQ-S tumors. (A) MTT (72 h) in HT29 vector and HT29 HLTF cells treated with the indicated combinations at 500 nM inhibitor +/− HCQ (10 µM). IGF1RAb, figitumumab; PTK2/FAKi, PF562271; PI3Ki, PF4691502. *P < 0.05. (B) HT29-vector and HT29-HLTF cells were grown as xenografts in nude mice. PBS or Lys05 (20 mg/kg) i.p. was adminstered daily. Daily tumor volumes mean +/− SEM. (C) Tumor volumes on the final day. (D) Tumor weights on the final day.

Article Snippet: For detection of RAD52 foci, HT29 cells were transfected with pCMV6 empty vector (Origene, PS100001) and pCMV6 HLTF (Origene, RC207920) and then the indicated cells were grown on chamber slides overnight to reach 50% confluence.

Techniques: Over Expression, Plasmid Preparation

Journal: Autophagy

Article Title: ALDH1A1 and HLTF modulate the activity of lysosomal autophagy inhibitors in cancer cells

doi: 10.1080/15548627.2017.1377377

Figure Lengend Snippet: HCQ-associated ROS produces DNA damage that is either repaired by HLTF-POLH, or results in DSB. (A) Reactive oxygen species (ROS) measured by flow cytometry. (B) Phosphorylation of H2AFX. Band quantification presented as mean +/− SD for multiple experiments. (C) DNA fragmentation is more striking in HLTF− than HLTF+ cells. (D) Two-wk colony formation assay in HT29 cells with stable expression of vector or HLTF: 2 mM NAC rescues HCQ (10 µM) cytotoxicity. (E) Tiron rescues HCQ-associated DNA damage. (F) Immunofluorescence microscopy demonstrates HLTF abrogates HCQ-induced RAD52+ double strand breaks (arrow). Sale bar 100 µm (G) Knockdown of DNA POLH. Quantification of bands: mean +/− SD from multiple experiments; 72-h MTT with siNT compared to siPOLH abrogates HCQ resistance due to HLTF overexpression in HT29 cells. (H) ATM inhibition with KU-60019 mitigates HLTF-mediated rescue of HCQ cytotoxicity in 72-h MTT assay of HT29 vector and HT29HLTF cells. *p < 0.05; ns, not significant..

Article Snippet: For detection of RAD52 foci, HT29 cells were transfected with pCMV6 empty vector (Origene, PS100001) and pCMV6 HLTF (Origene, RC207920) and then the indicated cells were grown on chamber slides overnight to reach 50% confluence.

Techniques: Flow Cytometry, Colony Assay, Expressing, Plasmid Preparation, Immunofluorescence, Microscopy, Over Expression, Inhibition, MTT Assay

Journal: Autophagy

Article Title: ALDH1A1 and HLTF modulate the activity of lysosomal autophagy inhibitors in cancer cells

doi: 10.1080/15548627.2017.1377377

Figure Lengend Snippet: Modulation of HCQ efficacy by ALDH1A1 and HLTF. (A) ROS levels in HCT15 and HT29 cells with overexpression or knockdown of ALHD1A1. (B) HCT15 cells transiently transfected with Control or ALDH1A1-expressing vector with or without HCQ (20 µM) for 12 h. (C) HT29 cells transfected with siNT (Non-Target) or siALDH1A1 with or without HCQ (20 µM) for 12 h. (D) Immunoblotting in lysates from the indicated cells treated with retinoic acid (RA 5 µM) +/− EZH2 inhibitor EPZ005687 2 µM (24 h). (E) Immunoblotting of lysates from HCT 15 cells transfected with control or ALDH1A1-expressing vector +/- EPZ005687 (2 µM), 24 h. (F) Immunoblotting from lysates from HCT15 cells treated with HCQ (20 µM) +/− Tiron (10 µM). (G) Immunoblotting of HCT15 cells transiently transfected with control or ALDH1A1-expressing vector +/− HCQ (20 µM) for 12 h. (H) ALDH1A1 facilitates entry of HCQ into the cell, where it accumulates in the lysosome. Lysosomal impairment leads to autophagy inhibition and the generation of ROS. ROS-associated DNA damage produces single-strand breaks with stalled replication forks. If HLTF is expressed, recruitment of the low fidelity DNA polymerase POLH allows translesion synthesis to occur promoting resistance to therapy. In the absence of HLTF-associated TLS, stalled replication forks collapse into double-strand breaks triggering cell death. Both RA and ROS can regulate KDM5D-PRC2-driven degradation of DNMT1 and upregulation of HLTF.

Article Snippet: For detection of RAD52 foci, HT29 cells were transfected with pCMV6 empty vector (Origene, PS100001) and pCMV6 HLTF (Origene, RC207920) and then the indicated cells were grown on chamber slides overnight to reach 50% confluence.

Techniques: Over Expression, Transfection, Expressing, Plasmid Preparation, Western Blot, Inhibition, Translesion Synthesis

Journal: BMC genomics

Article Title: Deciphering smooth muscle cell heterogeneity in atherosclerotic plaques and constructing model: a multi-omics approach with focus on KLF15/IGFBP4 axis.

doi: 10.1186/s12864-024-10379-y

Figure Lengend Snippet: Fig. 7 Model genes expression validation; GSEA enrichment analysis, and correlation analysis based on IGFBP4. The relative expression levels of IGFBP4, CTSC and APOE in A training and B testing sets. The relative expression levels of IGFBP4, CTSC and APOE in C GSE100927 and D GSE20129. E UpSet plot showing shared pathways in the GSEA enrichment analysis of early and advanced plaques from GES28829 and GSE43292, which based on the expression of IGFBP4. F The shared GSEA enrichment pathways based on the expression of IGFBP4. The scatterplot displaying the correlation between IGFBP4 and GACTA2, HLUM, ITAGLN. (*p < 0.05; **p < 0.01; ***p < 0.001****; p < 0.0001; ns, no statistical significance)

Article Snippet: Primary antibodies used included a rabbit polyclonal antibody against Igfbp4 (1:20 Cat No: 18500-1-AP, Proteintech) and a mouse monoclonal antibody against Tagln (1:500 Cat No: 60213-1-Ig, Proteintech).

Techniques: Expressing, Biomarker Discovery

Journal: BMC genomics

Article Title: Deciphering smooth muscle cell heterogeneity in atherosclerotic plaques and constructing model: a multi-omics approach with focus on KLF15/IGFBP4 axis.

doi: 10.1186/s12864-024-10379-y

Figure Lengend Snippet: Fig. 8 Pseudotime analysis between SMC2 and SMC5 clusters. A Nine stages of SMC differentiation. B The SMC2 and SMC5 cells using for pseudo-time analysis. C The temporal changing in cell differentiation. D The dynamic expression of IGFBP4 and marker genes along pseudotime

Article Snippet: Primary antibodies used included a rabbit polyclonal antibody against Igfbp4 (1:20 Cat No: 18500-1-AP, Proteintech) and a mouse monoclonal antibody against Tagln (1:500 Cat No: 60213-1-Ig, Proteintech).

Techniques: Cell Differentiation, Expressing, Marker

Journal: BMC genomics

Article Title: Deciphering smooth muscle cell heterogeneity in atherosclerotic plaques and constructing model: a multi-omics approach with focus on KLF15/IGFBP4 axis.

doi: 10.1186/s12864-024-10379-y

Figure Lengend Snippet: Fig. 9 Experimental validation of Igfbp4 expression. A Hematoxylin & eosin (HE) staining showing the macroscopic morphology and local magnification of vascular anatomical layers in rat carotid artery after balloon injury. (scale bar = 50 μm) B Localization of Igfbp4 expression in injury artery. HE Staining displaying the macroscopic and localized vascular landscapes of C normal artery, D early injured artery, and E advanced injured artery. (scale bar = 50 μm) F-G WB validating the protein expression levels of Igfbp4 in normal arteries, early injured arteries, and advanced injured arteries, using α-Tubulin as the housekeeping protein for normalization. H RT-qPCR experiment validating the mRNA expression levels of Igfbp4 in normal arteries, early injured arteries, and advanced injured arteries, using Gapdh as the housekeeping gene for normalization. (*p < 0.05; **p < 0.01; ***p < 0.001)

Article Snippet: Primary antibodies used included a rabbit polyclonal antibody against Igfbp4 (1:20 Cat No: 18500-1-AP, Proteintech) and a mouse monoclonal antibody against Tagln (1:500 Cat No: 60213-1-Ig, Proteintech).

Techniques: Biomarker Discovery, Expressing, Staining, Quantitative RT-PCR

Journal: BMC genomics

Article Title: Deciphering smooth muscle cell heterogeneity in atherosclerotic plaques and constructing model: a multi-omics approach with focus on KLF15/IGFBP4 axis.

doi: 10.1186/s12864-024-10379-y

Figure Lengend Snippet: Fig. 10 The prediction of TFs and targeted compound. A Network diagram of TFs binding with IGFBP4. B Box plot depicting the differences in TFs ex pression between early and advanced plaques. C The t-SNE plot displaying the expression level and localization of TFs in cell clusters. D The scatter plot showing the correlation between KLF15 and IGFBP4. E The enlarged t-SNE plot for KLF15. F The network diagram of compounds acting on KLF15. (*p < 0.05; **p < 0.01; ***p < 0.001)

Article Snippet: Primary antibodies used included a rabbit polyclonal antibody against Igfbp4 (1:20 Cat No: 18500-1-AP, Proteintech) and a mouse monoclonal antibody against Tagln (1:500 Cat No: 60213-1-Ig, Proteintech).

Techniques: Binding Assay, Expressing

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