Journal: Journal of Research in Medical Sciences : The Official Journal of Isfahan University of Medical Sciences

Article Title: Deciphering biological characteristics of tumorigenic subpopulations in human colorectal cancer reveals cellular plasticity

doi: 10.4103/1735-1995.187355

Figure Lengend Snippet: (a) Tumor cell size. (b) Complexity of intracellular structure of a tumor. (c) Sphere formation assay. (d) Aldehyde dehydrogenases enzyme activity of CD133 − cell subset isolated from primary colorectal cancer sample, (e) aldehyde dehydrogenases enzyme activity of CD133 − cell subset isolated from liver metastatic colorectal cancer sample, (f) aldehyde dehydrogenases enzyme activity of CD133 + cell subset isolated from primary colorectal cancer sample, and (g) aldehyde dehydrogenases enzyme activity of CD133 + cell subset isolated liver metastatic colorectal cancer sample

Article Snippet: Cell suspensions were incubated with a monoclonal CD133 antibody labeled with MicroBeads (Miltenyi Biotech) for 30 min at 4°C, and CD133 + cells were enriched using a MiniMACS magnet and MS columns (Miltenyi Biotech).

Techniques: Tube Formation Assay, Activity Assay, Isolation

Journal: Current protocols in protein science

Article Title: A Rapid and Facile Purification Method for Glycan-Binding Proteins and Glycoproteins.

doi: 10.1002/cpps.113

Figure Lengend Snippet: Figure 8 Separation of target-TCA complex from other components of the crude solution. The complex is separated from other impurities of the crude by centrifuging the mixture. The complex containing only the target and the TCA settles at the bottom of the microcentrifuge tube. The su- pernatant containing the remaining components of the crude extract is discarded. The precipitate is washed with buffer and then dissolved by competitive monovalent ligands. Thus the target cap- tured with TCA is released. The released target is separated from the TCA by filtration to get the target in pure form. Welch et al.

Article Snippet: SDS-PAGE running buffer (see recipe) SDS-PAGE sample buffer (see recipe) SDS-PAGE staining solution (see recipe) SDS-PAGE de-staining solution (see recipe) Welch et al. 22 of 27 Current Protocols in Protein Science TGX stain-free gels, 12% (BIO-RAD, cat. no. 4568043) Broad range prestained protein standard (New England Biolabs, cat. no. P7719S) β-Mercaptoethanol 0.6- and 1.5-ml microcentrifuge tubes Micropipets Micropipet tips Gel loading tips Mini-PROTEAN Tetra cell (BIO-RAD) PowerPac Power Supply (BIO-RAD) Microcentrifuge compatible heater (Themo Fisher Scientific) 1.

Techniques:

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: DHX9 contributes to the malignant phenotypes of colorectal cancer via activating NF-κB signaling pathway

doi: 10.1007/s00018-021-04013-3

Figure Lengend Snippet: DHX9 mediates the activation of NF-κB signal pathway in colorectal cancer cells. A Heatmap showed the alteration of gene expression (≥ twofold) obtained from RNA-Seq of vector-transfected and DHX9-silenced HCT116 cells. B Scatter plot displayed fold changes of gene expression (≥ twofold) in DHX9-silenced HCT116 cells compared with vector-transfected cells. C GO analysis of down-regulated genes (≥ twofold) in DHX9-silenced HCT116 cells compared with vector-transfected cells. D-E GSEA revealed that gene sets of cell motility D and NF-κB signaling E were enriched in DHX9-depleted HCT116 cells. F Proteins levels of IKBα, p65 and phosphorylated IKBα and p65 were evaluated by Western blotting in DHX9-overexpressed or -depleted HCT116 cells. G-H Cytosolic and nuclear fractionations were evaluated by Western blotting in DHX9-overexpressed HCT116 cells with or without p65 knockdown G or in DHX9-silenced HCT116 cells with or without LPS (1 μg/ml) pretreatment for 15 min H. I DHX9-overexpressed or -depleted HCT116 cells were subjected to immunofluorescence analysis of p65 localization (green). Vector-transfected (pLVX) HCT116 cells pretreated with LPS (1 μg/ml) for 15 min were served as a positive control. DAPI (blue) was applied to stain the nuclear. Scale bar, 20 μm. J HCT116 cells with stably expressing pLVX, pLVX-DHX9 or shRNAs against DHX9 were co-transfected with 0.5 μg NF-κB-TATA-Luc reporter plasmid and 10 ng Renilla luciferase reporter. 36 h after transfection, pLVX-HCT116 cells were treated with LPS (200 ng/mL) for 8 h. The luciferase activity of cells was measured. The values of firefly luciferase activity were normalized to Renilla luciferase activity. Fold activation were represented as mean ± SEM of three independent transfections. ***P < 0.001, one-way ANOVA with post hoc intergroup comparison by Tukey's test. K RT-qPCR experiment analyzed NF-κB-target genes including CXCL8, encoding IL-8; CCND1, encoding Cyclin D1; BIRC5, encoding Survivin; SNAI1, encoding Snail, in DHX9-overexpressed or -depleted HCT116 cells. Data were from three independent experiments and represented as mean ± SEM. ns, not significant; ***P < 0.001, one-way ANOVA with post hoc intergroup comparison by Tukey's test. L Western blotting analysis verified the protein levels of candidate NF-κB-target genes in DHX9-overexpressed or -depleted cells

Article Snippet: The cell lysates were sonicated to shear DNA to sizes of 200–500 bp and centrifuged at 12,000 g at 4 °C for 10 min. To preclear the chromatin, 60 µL of protein G agarose was added to each tube before immunoprecipitated with 5 µ g anti-p65 (Cat# 6956S, Cell Signaling Technology), anti-DHX9 (Cat# A300-855A, Invitrogen), anti-RNA pol II (Cat# A300-653A) or normal rabbit IgG (Cat# 2729S, Cell Signaling Technology) overnight at 4 °C with rotation.

Techniques: Activation Assay, Expressing, RNA Sequencing Assay, Plasmid Preparation, Transfection, Western Blot, Immunofluorescence, Positive Control, Staining, Stable Transfection, Luciferase, Activity Assay, Comparison, Quantitative RT-PCR

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: DHX9 contributes to the malignant phenotypes of colorectal cancer via activating NF-κB signaling pathway

doi: 10.1007/s00018-021-04013-3

Figure Lengend Snippet: DHX9 interacts with p65 and RNA Pol II to active NF-κB-mediated transcription in colorectal cancer cells. A-B Lysates of DHX9-overexpressed or -depleted HCT116 cells were subjected to immunoprecipitation with the anti-DHX9 or anti-IgG antibody, and then incubated with the indicated antibodies using Western blotting. C–D Lysates of DHX9-overexpressed or -depleted HCT116 cells were subjected to immunoprecipitation with the anti-p65 or anti-IgG antibody and subjected to Western blotting. E–G Stable HCT116 cells expressing WT DHX9 and K417R DHX9 were harvested and lysates were used to immunoprecipitation with the anti-DHX9 or anti-p65 or anti-IgG antibody, and then subjected to Western blotting. K417R indicates a negative mutant of DHX9, in which Lys 417 is substituted to Arg that abolishes the ATP-dependent helicase activity. H Stable HCT116 cells expressing WT DHX9 or K417R DHX9 were co-transfected with NF-κB-TATA-Luc reporter plasmid and Renilla luciferase reporter. The luciferase activity of cells was measured after 48-h transfection. The values of firefly luciferase activity were normalized to Renilla luciferase activity. Fold activation were represented as mean ± SEM of three independent transfections. ***P < 0.001, one-way ANOVA with post hoc intergroup comparison by Tukey's test

Article Snippet: The cell lysates were sonicated to shear DNA to sizes of 200–500 bp and centrifuged at 12,000 g at 4 °C for 10 min. To preclear the chromatin, 60 µL of protein G agarose was added to each tube before immunoprecipitated with 5 µ g anti-p65 (Cat# 6956S, Cell Signaling Technology), anti-DHX9 (Cat# A300-855A, Invitrogen), anti-RNA pol II (Cat# A300-653A) or normal rabbit IgG (Cat# 2729S, Cell Signaling Technology) overnight at 4 °C with rotation.

Techniques: Immunoprecipitation, Incubation, Western Blot, Expressing, Mutagenesis, Activity Assay, Transfection, Plasmid Preparation, Luciferase, Activation Assay, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: DHX9 contributes to the malignant phenotypes of colorectal cancer via activating NF-κB signaling pathway

doi: 10.1007/s00018-021-04013-3

Figure Lengend Snippet: DHX9 is required for p65 and RNA Pol II recruitment to the promoters of NF-κB-dependent genes. A–C ChIP-qPCR analysis for DHX9, p65 or RNA Pol II occupancy at the promoter of BIRC5 A, SNAI1 B or ACTB C in HCT116 cells stably expressed DHX9 cDNA or shDDR1 constructs. All data in bar graphs were shown as mean ± SEM from three independent experiments and analyzed by one-way ANOVA, post hoc intergroup comparisons, Tukey’s test. D A proposed working model of DHX9 in orchestrating the malignant phenotypes of CRC. On one hand, DHX9 enhances p65 phosphorylation, promotes p65 nuclear translocation to facilitate NF-κB-mediated transcriptional activity. On the other hand, DHX9 interacts with p65 and RNA Pol II and is necessary to recruit p65 and RNA Pol II to the NF-κB-dependent promoters to activate downstream gene transcription, including CXCL8, CCND1, BIRC5 and SNAI1. Enhanced CXCL8, Cyclin D1 and Survivin expression contributes to lower apoptosis and promote proliferation in CRC cells; the strengthened expression of Snail increases the capability of migration and invasion, and ultimately facilities liver colonization and metastasis in CRC

Article Snippet: The cell lysates were sonicated to shear DNA to sizes of 200–500 bp and centrifuged at 12,000 g at 4 °C for 10 min. To preclear the chromatin, 60 µL of protein G agarose was added to each tube before immunoprecipitated with 5 µ g anti-p65 (Cat# 6956S, Cell Signaling Technology), anti-DHX9 (Cat# A300-855A, Invitrogen), anti-RNA pol II (Cat# A300-653A) or normal rabbit IgG (Cat# 2729S, Cell Signaling Technology) overnight at 4 °C with rotation.

Techniques: Stable Transfection, Construct, Translocation Assay, Activity Assay, Expressing, Migration