Journal: Materials

Article Title: Effect of Hyaluronic Acid Content on Functional Properties, Antioxidant Activity, and In Vitro Digestion of Food-Grade Furcellaran Hydrogels and Emulgels

doi: 10.3390/ma18245581

Figure Lengend Snippet: ( a , b ) Effect of 2 h exposure to absorbed digestates from different composite gels on mitochondrial membrane potential in HT-29 cells. Data are presented as means ± SD of three independent repetitions. Statistically significant differences were investigated using one-way ANOVA followed by Tukey’s post hoc testing. Columns with the same letters are not significantly different ( p > 0.05). Staurosporine (1.5 µM) was used as a positive control. ( c ) Antioxidative potential of composite gels with different hyaluronic acid concentration before and after in vitro digestion.

Article Snippet: HT-29 human colon adenocarcinoma cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Membrane, Positive Control, Concentration Assay, In Vitro

Journal: Nature Communications

Article Title: Sequential transcriptional waves and NF-κB-driven chromatin remodeling direct drug-induced dedifferentiation in cancer

doi: 10.1038/s41467-026-71349-4

Figure Lengend Snippet: a Relative viability of DTPs versus DMSO controls for HCC827 cells treated with ERL and HT-29 treated with DAB + CET for 9 days. (mean ± SD, n = 3 independent measurements). b Heatmap of Log2 fold changes in expression of KDM5B, TGF-β receptor 2, and several TGF-β target genes at indicated treatment times relative to D0 in HCC827 (ERL) and HT-29 (DAB + CET) (heatmap is based on longitudinal RNA-seq datasets, n = 4 samples from 4 time points per cell line, with one biological replicate per time point). c Immunoblot of phospho(p)-SMAD2 (Ser465/467), SMAD2, p-c-Jun (Ser63), and c-Jun in HCC827 and HT-29 after 9 days of treatment as in ( a ) ( n = 3 independent experiments). d Gene set enrichment analysis (GSEA) showing negative enrichment of NRF2 pathway genes in HCC827 and HT-29 after 3 days of ERL or DAB + CET versus DMSO controls. Normalized enrichment score (NES) and false discovery rate (FDR) were assessed by GSEA using weighted enrichment statistics with gene-set permutation (1000 permutations). The analysis is based on the longitudinal RNA-seq dataset comparing the D3 drug-treated sample with the DMSO control sample ( n = 2 samples from 2 time points per cell line; one biological replicate per time point). e Change in cellular ROS levels in HCC827 and HT-29 upon 3 days of the indicated treatments relative to DMSO (E + N: ERL + NAC, D + C + N: DAB + CET + NAC, mean ± SD, two-sided Welch ANOVA test with Dunnett T3 correction for multiple comparisons, n = 3 independent measurements). Representative fields of view (FOV) of RelA immunofluorescence staining in HCC827 ( f ) and HT-29 ( g ) under the indicated conditions ( n = 3 independent experiments, scale bar: 50 μm). Quantitation of nuclear RelA fluorescence intensity in HCC827 ( h ) and HT-29 ( i ) (two-sided Kruskal-Wallis test with Dunn’s correction for multiple comparisons; center line, box limits, and whiskers denote median, 25th/75th percentiles, and 10th/90th percentiles, respectively; n = 496, 568, 568 single cells from randomly selected FOVs for control, ERL, and E + N, and n = 690, 690, 690 for control, DAB + CET, and D + C + N). ChIP-qPCR of RelA binding at promoter regions of canonical RelA target genes in HCC827 ( j ) and HT-29 ( k ) cells after 3 days of treatment versus DMSO (mean ± SD, n = 3 independent measurements). Relative viability of HCC827 ( l ) and HT-29 ( m ) treated with ERL or DAB + CET alone or in combination with JSH-23; treatment was stopped upon clear regrowth in the ERL-alone and DAB + CET-only groups, and cell numbers were assessed by nuclear staining and counting (mean ± SD, n = 9 randomly selected FOVs from 3 independent experiments). n Relative viability of HCC827, HT-29, M397, and M229 after 3 days of the indicated treatments, assessed by nuclear staining and counting (mean ± SD, n = 9 randomly selected FOVs from 3 independent experiments). For a , j – n , see Statistics analysis in “Methods” for the statistical test used. Source data are provided as a file.

Article Snippet: HCC827 (CRL-2868) and HT-29 (HTB-38) were purchased from ATCC and cultured in ATCC-formulated RPMI-1640 Medium (ATCC 30-2001) and ATCC-formulated McCoy’s 5a Medium Modified (ATCC 30-2007), respectively, in a water-saturated incubator at 37 °C with 5% CO 2 .For long-term induction of dedifferentiation towards a DTP state, HCC827 cells were treated with 2 μM ERL, and HT-29 cells were treated with 50 μg/mL cetuximab (CET, Selleckchem A2000) in combination with 1 μM DAB for 3 weeks.

Techniques: Expressing, RNA Sequencing, Western Blot, Control, Immunofluorescence, Staining, Quantitation Assay, Fluorescence, ChIP-qPCR, Binding 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 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, 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: 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: 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: 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

Journal: Renal Failure

Article Title: IGFBP2 and IGFBP4 interact to activate complement pathway in diabetic kidney disease

doi: 10.1080/0886022x.2024.2440528

Figure Lengend Snippet: Figure 1. The proteomic analysis of plasma was conducted across three groups: normal adults, diabetic patients, and patients with diabetic kidney disease. (A) A Venn diagram was utilized to analyze the sequenced proteins, where group D represents the plasma samples from diabetic patients, group DK denotes the plasma from DKD patients, and group nor corresponds to the plasma of normal adults. The numbers indicate the count of sequenced and annotated proteins. (B) Heat maps were generated to analyze the differential expression of plasma proteins in patients with diabetes and those with diabetic kidney disease. (C) A volcano plot illustrated the significant differences in plasma proteins between diabetic patients and those with DKD. (D) An analysis of protein–protein interactions highlighted significant differences in plasma proteins among patients with diabetes and diabetic kidney disease. (E) The analysis focused on plasma-specific proteins and their interactions in patients with DKD. (F, G) Investigated the enrichment of plasma-specific protein Gene Ontology (GO) and KEGG database pathways in patients with diabetic kidney disease. (H) The plasma levels of IGFBP2 and IGFBP4 in normal adults, diabetic patients, and diabetic kidney patients were quantified using Western blotting (WB). Data are presented as mean ± SD (n = 3). **p < .01.

Article Snippet: Primary antibodies were then added, including IGFbP4 (Pa5-25925, thermo Fisher, Waltham, Ma), IGFbP2 (Pa5-79450, thermo Fisher, Waltham, Ma), C3 (Pa5-21349, thermo Fisher, Waltham, Ma), C4b (22233-1-aP, Proteintech, Wuhan, China), C5 (ab275931, abcam, Cambridge, uK), C9 (ab168345, abcam, Cambridge, uK), GaPdH (ab8245, abcam, Cambridge, uK), and transferrin (Pa5-27306, thermo Fisher, Waltham, Ma). these were incubated overnight at 4 °C, followed by washing with tbSt buffer. the samples were then combined with Goat anti-Mouse IgG/HrP (SE131, Solarbio, beijing, China) and Goat anti-rabbit IgG H&L (HrP) (ab6721, abcam, Cambridge, uK), and incubated at room temperature for 2 h. ECL color development was performed, and gray values were measured using an automatic gel imaging analyzer.

Techniques: Clinical Proteomics, Generated, Quantitative Proteomics, Protein-Protein interactions, Western Blot

Journal: Renal Failure

Article Title: IGFBP2 and IGFBP4 interact to activate complement pathway in diabetic kidney disease

doi: 10.1080/0886022x.2024.2440528

Figure Lengend Snippet: Figure 2. IGFBP2 and IGFBP4 contribute to the progression of DKD in mice. (A) Enzyme-linked immunosorbent assay (ELISA) was employed to assess the levels of serum albumin, fasting glucose, and serum creatinine in both normal mice and diabetic mice with nephropathy. The CK group consisted of normal mice, while the DK group comprised diabetic mice with nephropathy. (B) Insulin tolerance tests were conducted on normal mice and mice with DKD, and the area under the curve for the test data was calculated. (C) ELISA was utilized to evaluate the changes in urinary albumin, urinary creatinine, and urinary glucose between normal mice and those with DKD. (D) Hematoxylin and eosin (HE) staining was performed to observe the pathological alterations in kidney tissue from normal mice and those with DKD, while periodic acid-Schiff (PAS) staining was used to assess glycogen deposition, and Masson’s tri chrome staining was applied to examine collagen deposition in the kidney tissue. Scale bar: 50 µm. (E) The expression levels of IGFBP2 and IGFBP4 proteins in the plasma of normal mice and those with DKD were analyzed via Western blotting (WB) from the second to the sixth week. (F) Correlation analyses were conducted to evaluate the relationship between plasma IGFBP2 and IGFBP4 expression levels and serum albumin, fasting glucose, and serum creat inine in DKD mice. Data are presented as mean ± SD. n = 3. *p < .05, **p < .01, ***p < .001, compared to CK.

Article Snippet: Primary antibodies were then added, including IGFbP4 (Pa5-25925, thermo Fisher, Waltham, Ma), IGFbP2 (Pa5-79450, thermo Fisher, Waltham, Ma), C3 (Pa5-21349, thermo Fisher, Waltham, Ma), C4b (22233-1-aP, Proteintech, Wuhan, China), C5 (ab275931, abcam, Cambridge, uK), C9 (ab168345, abcam, Cambridge, uK), GaPdH (ab8245, abcam, Cambridge, uK), and transferrin (Pa5-27306, thermo Fisher, Waltham, Ma). these were incubated overnight at 4 °C, followed by washing with tbSt buffer. the samples were then combined with Goat anti-Mouse IgG/HrP (SE131, Solarbio, beijing, China) and Goat anti-rabbit IgG H&L (HrP) (ab6721, abcam, Cambridge, uK), and incubated at room temperature for 2 h. ECL color development was performed, and gray values were measured using an automatic gel imaging analyzer.

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Expressing, Clinical Proteomics, Western Blot

Journal: Renal Failure

Article Title: IGFBP2 and IGFBP4 interact to activate complement pathway in diabetic kidney disease

doi: 10.1080/0886022x.2024.2440528

Figure Lengend Snippet: Figure 3. The effects of IGFBP2 and IGFBP4 on the complement pathway in DKD mice were examined. (A) The alterations in IGFBP2, IGFBP4, and comple ment proteins C3, C4B, C5, and C9 in the renal tissues of both normal and DKD mice were assessed using immunohistochemistry, with a scale bar of 50 μm. (B) The levels of plasma IGFBP2, IGFBP4, and complement proteins C3, C4B, C5, and C9 in normal and DKD mice were analyzed via Western blotting (WB). (C) The changes in IGFBP2, IGFBP4, and complement proteins C3, C4B, C5, and C9 in the renal tissues of normal and DKD mice were also evaluated using WB. (D) Plasma levels of MAC and MBL were measured using ELISA in both normal and DKD mice. Data are presented as mean ± SD, with n = 3. Statistical significance is indicated as **p < .01 and ***p < .001.

Article Snippet: Primary antibodies were then added, including IGFbP4 (Pa5-25925, thermo Fisher, Waltham, Ma), IGFbP2 (Pa5-79450, thermo Fisher, Waltham, Ma), C3 (Pa5-21349, thermo Fisher, Waltham, Ma), C4b (22233-1-aP, Proteintech, Wuhan, China), C5 (ab275931, abcam, Cambridge, uK), C9 (ab168345, abcam, Cambridge, uK), GaPdH (ab8245, abcam, Cambridge, uK), and transferrin (Pa5-27306, thermo Fisher, Waltham, Ma). these were incubated overnight at 4 °C, followed by washing with tbSt buffer. the samples were then combined with Goat anti-Mouse IgG/HrP (SE131, Solarbio, beijing, China) and Goat anti-rabbit IgG H&L (HrP) (ab6721, abcam, Cambridge, uK), and incubated at room temperature for 2 h. ECL color development was performed, and gray values were measured using an automatic gel imaging analyzer.

Techniques: Immunohistochemistry, Clinical Proteomics, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Renal Failure

Article Title: IGFBP2 and IGFBP4 interact to activate complement pathway in diabetic kidney disease

doi: 10.1080/0886022x.2024.2440528

Figure Lengend Snippet: Figure 4. There is a protein interaction between IGFBP2 and IGFBP4. (A) IGFBP2 was overexpressed in 293T cells, and the expression level of IGFBP2 was assessed using qPCR to confirm the successful construction of the model. The group with overexpressed IGFBP2 is referred to as oe-IGFBP2. (B) Western blotting (WB) was employed to measure the levels of IGFBP2 and IGFBP4 following the overexpression of IGFBP2 in 293T cells. (C) Co-immunoprecipitation (Co-IP) was utilized to detect the formation of the IGFBP2 and IGFBP4 complex. Data are presented as mean ± SD, with n = 3 and **p < .01.

Article Snippet: Primary antibodies were then added, including IGFbP4 (Pa5-25925, thermo Fisher, Waltham, Ma), IGFbP2 (Pa5-79450, thermo Fisher, Waltham, Ma), C3 (Pa5-21349, thermo Fisher, Waltham, Ma), C4b (22233-1-aP, Proteintech, Wuhan, China), C5 (ab275931, abcam, Cambridge, uK), C9 (ab168345, abcam, Cambridge, uK), GaPdH (ab8245, abcam, Cambridge, uK), and transferrin (Pa5-27306, thermo Fisher, Waltham, Ma). these were incubated overnight at 4 °C, followed by washing with tbSt buffer. the samples were then combined with Goat anti-Mouse IgG/HrP (SE131, Solarbio, beijing, China) and Goat anti-rabbit IgG H&L (HrP) (ab6721, abcam, Cambridge, uK), and incubated at room temperature for 2 h. ECL color development was performed, and gray values were measured using an automatic gel imaging analyzer.

Techniques: Expressing, Western Blot, Over Expression, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Renal Failure

Article Title: IGFBP2 and IGFBP4 interact to activate complement pathway in diabetic kidney disease

doi: 10.1080/0886022x.2024.2440528

Figure Lengend Snippet: Figure 5. The crosstalk effect among HK-2 cells, THP-1 cells, and primary human renal podocytes was investigated as follows: (A) the serum from DKD mice (10%) was added to HK-2 cells for culture for 24 h, after which relevant indicators were detected. The supernatant from HK-2 cells was then transferred to THP-1 cells to assess the functional changes in THP-1 cells, and subsequently, the THP-1 cell supernatant was added to primary human renal podocytes to observe their functional alterations. (B) The levels of IGFBP2 and IGFBP4 following HK-2 cell stimulation were measured. (C) The polarization of THP-1 cells was assessed using flow cytometry, where CD86% served as the marker for M1 polarization and CD206% indicated M2 polarization. (D) ELISA was employed to detect changes in complement proteins C3, C4B, C5, and C9 in THP-1 cells. (E) The levels of MAC and MBL in THP-1 cells were also measured using ELISA. (F) Podocyte proliferation was evaluated using the CCK-8 assay. (G) Apoptosis of primary human renal podocytes was analyzed through flow cytometry. (H) The expression of reactive oxygen species (ROS) in primary human renal podocytes was visualized by immunofluorescence, with green fluorescence indicating the intensity of ROS expression. Data are presented as mean ± SD (n = 3; *p < .05, **p < .01, ***p < .001).

Article Snippet: Primary antibodies were then added, including IGFbP4 (Pa5-25925, thermo Fisher, Waltham, Ma), IGFbP2 (Pa5-79450, thermo Fisher, Waltham, Ma), C3 (Pa5-21349, thermo Fisher, Waltham, Ma), C4b (22233-1-aP, Proteintech, Wuhan, China), C5 (ab275931, abcam, Cambridge, uK), C9 (ab168345, abcam, Cambridge, uK), GaPdH (ab8245, abcam, Cambridge, uK), and transferrin (Pa5-27306, thermo Fisher, Waltham, Ma). these were incubated overnight at 4 °C, followed by washing with tbSt buffer. the samples were then combined with Goat anti-Mouse IgG/HrP (SE131, Solarbio, beijing, China) and Goat anti-rabbit IgG H&L (HrP) (ab6721, abcam, Cambridge, uK), and incubated at room temperature for 2 h. ECL color development was performed, and gray values were measured using an automatic gel imaging analyzer.

Techniques: Functional Assay, Cell Stimulation, Flow Cytometry, Marker, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Expressing, Immunofluorescence, Fluorescence

Journal: Renal Failure

Article Title: IGFBP2 and IGFBP4 interact to activate complement pathway in diabetic kidney disease

doi: 10.1080/0886022x.2024.2440528

Figure Lengend Snippet: Figure 6. Effects of exogenous addition of IGFBP2 and IGFBP4 on THP-1 cells and primary human renal podocytes. (A) Following the addition of recombi nant IGFBP2 and IGFBP4 proteins to THP-1 cells, the supernatant was collected for the assessment of related indices, which were subsequently applied to primary human renal podocytes to observe functional changes. (B) Different concentrations of recombinant IGFBP2 and IGFBP4 (50 ng/mL, 100 ng/mL, 200 ng/mL, and 400 ng/mL) were found to stimulate the levels of complement proteins C3, C4B, C5, C9, as well as MAC and MBL in THP-1 cells. (C) The effects of individually adding IGFBP2h or IGFBP4 recombinant protein, or their combined addition, on the levels of complement proteins C3, C4B, C5, C9, MAC, and MBL in THP-1 cells were assessed. (D) Flow cytometry analysis was conducted to determine the impact of IGFBP2h or IGFBP4 recombinant protein, either alone or in combination, on the polarization of THP-1 cells. (E) The influence of THP-1 cell supernatant on podocyte proliferation was eval uated using the CCK-8 assay. (F) The effect of THP-1 cell supernatant on podocyte apoptosis was measured through flow cytometry. (G) Immunofluorescence was utilized to observe the effect of THP-1 cell supernatant on ROS expression in primary human renal podocytes. (H) The impact of THP-1 cell supernatant on podocyte morphology was also assessed. Data are presented as mean ± SD. n = 3; *p < .05, **p < .01, ***p < .001.

Article Snippet: Primary antibodies were then added, including IGFbP4 (Pa5-25925, thermo Fisher, Waltham, Ma), IGFbP2 (Pa5-79450, thermo Fisher, Waltham, Ma), C3 (Pa5-21349, thermo Fisher, Waltham, Ma), C4b (22233-1-aP, Proteintech, Wuhan, China), C5 (ab275931, abcam, Cambridge, uK), C9 (ab168345, abcam, Cambridge, uK), GaPdH (ab8245, abcam, Cambridge, uK), and transferrin (Pa5-27306, thermo Fisher, Waltham, Ma). these were incubated overnight at 4 °C, followed by washing with tbSt buffer. the samples were then combined with Goat anti-Mouse IgG/HrP (SE131, Solarbio, beijing, China) and Goat anti-rabbit IgG H&L (HrP) (ab6721, abcam, Cambridge, uK), and incubated at room temperature for 2 h. ECL color development was performed, and gray values were measured using an automatic gel imaging analyzer.

Techniques: Functional Assay, Recombinant, Flow Cytometry, CCK-8 Assay, Immunofluorescence, Expressing