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: 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: Cell Death and Differentiation

Article Title: DUB3 deubiquitinates and stabilizes NRF2 in chemotherapy resistance of colorectal cancer

doi: 10.1038/s41418-019-0303-z

Figure Lengend Snippet: DUB3 is a potential DUB of NRF2 and correlated expressed with NRF2 in colon cancer. a DUB3 has the strongest effect on NRF2 protein level among the top 10 DUBs. HEK293T cells were cotransfected with NRF2-Myc and individual top 10 DUBs-Flag overexpresiong vectors. Forty-eight hours after transfection, the cell lysates were analyzed by western blot with the indicated antibodies. NRF2-Myc protein expression was normalized to GAPDH. b DUB3 has the strongest effect on NRF2 transcriptional activity among the top 10 DUBs. HEK293T cells were cotransfected with ARE reporter firefly luciferase (100 ng), pRL-TK (10 ng) and the indicated amounts of DUBs. Reporter assays were performed 24 h after transfection, and the results are presented as the NRF2/TK luciferase activity. c Oncomine data mining analysis of NRF2 mRNA levels in three different datasets comparing normal tissues vs. CRC tissues. d The protein expression of NRF2 and DUB3 in 24 representative pairs of primary CRC (T) and adjacent non-tumor tissues (N). e The protein expression of NRF2 and DUB3 in the human embryonic kidney cell line HEK293T, the normal human colon cell line FHC and seven colorectal cell lines (HCT116, SW48, SW480, RKO, DLD1, LOVO, and HT29)

Article Snippet: Cell culture Human CRC cell lines including HCT116, SW48, SW480, RKO, DLD1, LOVO, HT29, the human normal colon cell line FHC, the human embryonic kidney 293T cell line (HEK293T), and the human osteosarcoma cell line U2OS were purchased from China Center for Type Culture Collection (CCTCC).

Techniques: Transfection, Western Blot, Expressing, Activity Assay, Luciferase