Journal: Molecular and Cellular Biology

Article Title: Hypoxic Environment Promotes Barrier Formation in Human Intestinal Epithelial Cells through Regulation of MicroRNA 320a Expression

doi: 10.1128/MCB.00553-18

Figure Lengend Snippet: Hypoxia improves barrier function in intestinal epithelial cells. T84 cells were seeded onto Transwell inserts and cultured for the indicated time under normoxic (21% O2) (red) or hypoxic conditions (1% O2) (blue). (A) The rate of TEER increase over the cell monolayer was measured every 24 h using the EVOM2 chopstick electrode. A TEER of >330 Ω ⋅ cm2 indicates complete barrier formation and is marked by a dotted line (36). (B) Paracellular permeability of the cell monolayer on Transwell inserts was assessed by adding 4-kDa FITC-dextran to the apical compartment (schematic overview [left panel]). At 3 h postincubation, the basal medium was analyzed for an increase of fluorescence by spectrofluorimetry (right panel). (C) T84 cells cultured for 24 and 48 h under normoxic and hypoxic conditions were evaluated for the expression of the tight junction protein ZO-1 (red). Cell nuclei were stained with DAPI (blue). Scale bar, 25 μm. A representative image is shown. (D) RNA samples of normoxic and hypoxic cultures of T84 cells taken at 24-h intervals for 4 days were analyzed by qPCR for the expression of the hypoxia-induced VEGF and CA9 genes. The results are normalized to normoxic values for each day. (E) RNA samples of normoxic and hypoxic cultures of T84 cells were analyzed over time by qPCR for the expression of the tight junction proteins E-cadherin, occludin, and JAM-A. The results are normalized to normoxic values for each day. (A and B) Values shown represent means ± the standard errors of the mean (SEM) (n = 9) from triplicate experiments. ***, P < 0.0001 (two-way analysis of variance [ANOVA]). (D and E) Experiments were performed in quadruplicate. Error bars indicate the standard deviations (SD). *, P < 0.05; **, < 0.01; n.s., not significant (one-sample t test on log-transformed fold changes).

Article Snippet: T84 human colonic adenocarcinoma cells (ATCC CCL-248) were cultured in Gibco Dulbecco modified Eagle medium–F-12 nutrient mixture (1:1), supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco) in collagen-coated T25 cell culture flasks.

Techniques: Cell Culture, Permeability, Fluorescence, Expressing, Staining, Transformation Assay

Journal: Molecular and Cellular Biology

Article Title: Hypoxic Environment Promotes Barrier Formation in Human Intestinal Epithelial Cells through Regulation of MicroRNA 320a Expression

doi: 10.1128/MCB.00553-18

Figure Lengend Snippet: Chemical stabilization of HIF by DMOG to mimic hypoxia induces a faster barrier formation. (A) Schematic showing the regulation of the transcription factor HIF-1α/2α at high and low oxygen concentrations. Under normoxic conditions, HIF-1α/2α is hydroxylated at two specific proline residues by different prolyl hydroxylases (PHDs), leading to binding to the E3 ubiquitin ligase containing the von Hippel-Lindau (VHL) tumor suppressor protein. This mediates the polyubiquitination of HIF-1α/2α and its downstream proteasomal degradation. Under hypoxic conditions, degradation is inhibited due to the lack of substrate for the PHDs, therefore stabilizing HIF-1α/2α, leading to dimerization with its constitutively expressed β-subunit (HIF-1β) and subsequent gene expression. The pharmacological activation of HIF-1-function by DMOG and inhibition by shRNA against HIF mRNA are shown. (B) T84 cells were incubated in normoxic (N) or hypoxic (H) conditions for 6 h or treated with dimethyl sulfoxide (DMSO) or DMOG for 6 h. Protein was harvested, and Western blotting was performed for HIF-1α and HIF-2α. Actin was used as a loading control. Each experiment was performed in triplicate. A representative image is shown. (C) T84 cells were seeded and incubated under normoxic conditions in the presence or absence of DMOG. DMSO was used as a solvent control. RNA was isolated, and the upregulation of VEGF and CA9 was evaluated by qPCR. The results are normalized to untreated cells. (D) T84 cells were seeded on Transwell inserts and incubated under normoxic conditions in the presence or absence of DMOG. DMSO was used as a solvent control. TEER measurements were taken in 24-h intervals for 4 days. A TEER of >330 Ω ⋅ cm2 indicates complete barrier formation and is marked by a dashed line. The values shown represent means ± the SEM (n = 9) from triplicate experiments. (C) *, P < 0.05; ***, P < 0.001 (one-sample t test on log-transformed fold changes). (D) *, P = 0.0417 (two-way ANOVA).

Article Snippet: T84 human colonic adenocarcinoma cells (ATCC CCL-248) were cultured in Gibco Dulbecco modified Eagle medium–F-12 nutrient mixture (1:1), supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco) in collagen-coated T25 cell culture flasks.

Techniques: Binding Assay, Ubiquitin Proteomics, Gene Expression, Activation Assay, Inhibition, shRNA, Incubation, Western Blot, Control, Solvent, Isolation, Transformation Assay

Journal: Molecular and Cellular Biology

Article Title: Hypoxic Environment Promotes Barrier Formation in Human Intestinal Epithelial Cells through Regulation of MicroRNA 320a Expression

doi: 10.1128/MCB.00553-18

Figure Lengend Snippet: HIF-1α is responsible for faster barrier establishment under hypoxic conditions. T84 cells were transduced with lentiviruses expressing an shRNA targeting HIF-1α or a scrambled control. (A) Knockdown of HIF-1α was controlled by qPCR. (B) T84 cells knocked down for HIF-1α or expressing a scrambled control were incubated under normoxic or hypoxic conditions. At 6 h postincubation, protein samples were harvested and evaluated by Western blotting for HIF-1α or HIF-2α. Each experiment was performed in triplicate; a representative image is shown. (C) T84 cells expressing a scramble shRNA or a shRNA against HIF-1α were evaluated for their induction of the hypoxic marker CA9 over the indicated time course by qPCR. The results are normalized to normoxic values for each day. (D) T84 cells expressing a scramble shRNA or a shRNA against HIF-1α were evaluated for their induction of tight junction proteins over the indicated time course by qPCR. The results are normalized to normoxic values for each day. (E) T84 cells expressing a scramble shRNA or a shRNA against HIF-1α or HIF-2α were seeded on Transwell inserts. Cells were incubated in normoxic or hypoxic conditions, and TEER measurements were taken at 24-h intervals for 5 days. A TEER of >330 Ω ⋅ cm2 indicates complete barrier formation and is marked by a dotted line. Values shown represent means ± the SEM (n = 9) from triplicate experiments for shScrambled and shHIF-1α. For shHIF-2α, one representative experiment is shown of two different anti-HIF-2α shRNAs tested. *, P < 0.05; n.s., not significant (two-way ANOVA). (A, C, and D) Values shown represent the means plus the SD from three independent experiments (***, P < 0.001; one-sample t test on log-transformed fold changes).

Article Snippet: T84 human colonic adenocarcinoma cells (ATCC CCL-248) were cultured in Gibco Dulbecco modified Eagle medium–F-12 nutrient mixture (1:1), supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco) in collagen-coated T25 cell culture flasks.

Techniques: Transduction, Expressing, shRNA, Control, Knockdown, Incubation, Western Blot, Marker, Transformation Assay

Journal: Molecular and Cellular Biology

Article Title: Hypoxic Environment Promotes Barrier Formation in Human Intestinal Epithelial Cells through Regulation of MicroRNA 320a Expression

doi: 10.1128/MCB.00553-18

Figure Lengend Snippet: Hypoxia leads to changes in expression of several hypoxamiRs known to regulate barrier function. T84 cells were incubated under hypoxic or normoxic conditions for 48 h. miRNA was isolated and evaluated by miRNA microarray. (A and B) Heatmaps of differentially expressed miRNAs in T84 cells cultured under normoxic and hypoxic conditions. The color scale shown on the right illustrates the relative expression levels of differentially expressed miRNAs. Orange indicates upregulated miRNAs (>0), and purple indicates downregulated miRNAs (<0). (A) Heatmap for 108 differentially regulated hypoxamiRs that were significantly up- or downregulated compared to normoxic conditions. Connecting lines in the cluster dendrogram between up- and downregulated miRNAs were shortened to enable visualization (indicated by two skewed lines). (B) Heatmap of miRNA-210-3p (positive control for hypoxic conditions), miRNA-320a, miRNA-34a-5p, and miRNA16-5p, identified by pathway analysis for playing a putative role in barrier formation. For both panels A and B, samples were tested in quadruplicate, and the level of expression of each replicate is shown in the heatmap.

Article Snippet: T84 human colonic adenocarcinoma cells (ATCC CCL-248) were cultured in Gibco Dulbecco modified Eagle medium–F-12 nutrient mixture (1:1), supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco) in collagen-coated T25 cell culture flasks.

Techniques: Expressing, Incubation, Isolation, Microarray, Cell Culture, Positive Control

Journal: Molecular and Cellular Biology

Article Title: Hypoxic Environment Promotes Barrier Formation in Human Intestinal Epithelial Cells through Regulation of MicroRNA 320a Expression

doi: 10.1128/MCB.00553-18

Figure Lengend Snippet: Validation of upregulated hypoxamiRs in T84 cells and primary human mini-gut organoids. T84 (A) and human intestinal epithelial cells (hIEC) cultured as mini-gut organoids (B) were incubated for 24 and 48 h under hypoxic or normoxic conditions. Upregulation of specific miRNAs was evaluated by qRT-PCR. miRNA-210-3p was used as a positive control for a miRNA upregulated under hypoxia. miRNA-1268a was used as a negative control since its expression is not hypoxia dependent. Data were normalized to normoxic cells at 24 h. All experiments were performed in triplicate. Error bars indicate the SEM.

Article Snippet: T84 human colonic adenocarcinoma cells (ATCC CCL-248) were cultured in Gibco Dulbecco modified Eagle medium–F-12 nutrient mixture (1:1), supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco) in collagen-coated T25 cell culture flasks.

Techniques: Biomarker Discovery, Cell Culture, Incubation, Quantitative RT-PCR, Positive Control, Negative Control, Expressing

Journal: Molecular and Cellular Biology

Article Title: Hypoxic Environment Promotes Barrier Formation in Human Intestinal Epithelial Cells through Regulation of MicroRNA 320a Expression

doi: 10.1128/MCB.00553-18

Figure Lengend Snippet: Overexpression of miRNA-320a induces faster barrier formation in T84 cells. (A) T84 cells stably overexpressing miRNA-320a, miRNA-16-5p, and miRNA-34a-5p by lentiviral transduction were seeded onto Transwell inserts, and barrier formation was assessed by TEER measurement in 24-h intervals over 4 days. A TEER of >330 Ω ⋅ cm2 indicates complete barrier formation and is marked by a dotted line. Values shown represent means ± the SEM (n = 6 [miRNA-16-5p and miRNA-34a-5p] or n = 12 [miRNA-320a]) from triplicate or quadruplicate experiments, respectively. ***, P = 0.0002 (two-way ANOVA); n.s., not significant. (B) T84 cells overexpressing miR-320a, miR-16-5p, and miR-34a-5p were harvested, and the overexpression of each miRNA was controlled by qRT-PCR. Values shown represent means ± the SD from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (one-sample t test on log-transformed fold changes).

Article Snippet: T84 human colonic adenocarcinoma cells (ATCC CCL-248) were cultured in Gibco Dulbecco modified Eagle medium–F-12 nutrient mixture (1:1), supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco) in collagen-coated T25 cell culture flasks.

Techniques: Over Expression, Stable Transfection, Transduction, Quantitative RT-PCR, Transformation Assay

Journal: Molecular and Cellular Biology

Article Title: Hypoxic Environment Promotes Barrier Formation in Human Intestinal Epithelial Cells through Regulation of MicroRNA 320a Expression

doi: 10.1128/MCB.00553-18

Figure Lengend Snippet: Inhibition of miRNA-320a expression diminishes barrier formation in T84 cells. (A) T84 cells stably expressing miRNA-320a sponge were seeded onto Transwell inserts under normoxic or hypoxic conditions and barrier function was assessed by TEER measurements in 24-h intervals over 4 days. A TEER of >330 Ω ⋅ cm2 indicates complete barrier formation and is marked by a dotted line. Values shown represent means ± the SEM (n = 12) from quadruplicate experiments. *, P = 0.0174 (two-way ANOVA). (B) Paracellular permeability of T84 cells overexpressing the miRNA-320a (overexpression [OE]) or the miRNA-320a sponge. The barrier function on Transwell inserts was assessed by adding 4 kDa FITC-dextran to the apical compartment and measuring the diffusion of fluorescent FITC-dextran to the basal compartment at 3 h posttreatment. A paracellular permeability assay was performed every 24 h for 4 days. Values shown represent the means ± the SEM (n = 3) from triplicate experiments. (C) T84 cells overexpressing (OE) miR-320a or depleted of miR-320a by expression of a sponge were evaluated by qRT-PCR. Values shown represent means ± the SD from three independent experiments. **, P < 0.01; ***, P < 0.001 (one-sample t test on log-transformed fold changes).

Article Snippet: T84 human colonic adenocarcinoma cells (ATCC CCL-248) were cultured in Gibco Dulbecco modified Eagle medium–F-12 nutrient mixture (1:1), supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco) in collagen-coated T25 cell culture flasks.

Techniques: Inhibition, Expressing, Stable Transfection, Permeability, Over Expression, Diffusion-based Assay, Quantitative RT-PCR, Transformation Assay

Journal: Molecular and Cellular Biology

Article Title: Hypoxic Environment Promotes Barrier Formation in Human Intestinal Epithelial Cells through Regulation of MicroRNA 320a Expression

doi: 10.1128/MCB.00553-18

Figure Lengend Snippet: HIF-1α binds to the promoter region of miRNA-320a and controls its expression. (A) HIF-1α binding sites around the miRNA-320a genomic region were analyzed from ChIP-seq data sets of breast cancer T47D (light blue) and HeLa cells (dark blue) cultured under hypoxic conditions. Part of chromosome 8 which contains the coding sequence for miRNA-320a is displayed. (B) T84 cells expressing a scrambled shRNA or a shRNA directed against HIF-1α were incubated under normoxic or hypoxic conditions. At 24 and 48 h postincubation, the levels of miRNA-320a were analyzed by qRT-PCR. Experiments were performed in triplicate; error bars indicate means ± the SEM.

Article Snippet: T84 human colonic adenocarcinoma cells (ATCC CCL-248) were cultured in Gibco Dulbecco modified Eagle medium–F-12 nutrient mixture (1:1), supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco) in collagen-coated T25 cell culture flasks.

Techniques: Expressing, Binding Assay, ChIP-sequencing, Cell Culture, Sequencing, shRNA, Incubation, Quantitative RT-PCR

Journal: Medical Journal of the Islamic Republic of Iran

Article Title: Human papilloma virus: A review study of epidemiology, carcinogenesis, diagnostic methods, and treatment of all HPV-related cancers

doi: 10.47176/mjiri.35.65

Figure Lengend Snippet: The classification of commercially available diagnostic techniques for the detection and genotyping of HPV

Article Snippet: , , PCR-based fluorescent bead array , ❖ Microarray-based HPV genotyping assays ○ The PapilloCheck HPV Screening Test(greiner bio), ○ Clart HPV 2 – papillomavirus clinical arrays( Genomica), ○ HPV GenoArray Test Kit ( Hybribio), ○ GeneTrack HPV DNA Chip( Daejeon), ○ GeneSQUARE HPV Microarray,( Kurabo) ○ INFINITI HPV-HR QUAD (AutoGenomics), ○ PANArray HPV Genotyping Chip( Panagene), ○ GG HPVCHIP ( Goodgene) ❖ Suspension array based HPV genotyping assays ○ The Multiplex HPV Genotyping Kit (Multimetrix, Heidelberg) ○ digene HPV Genotyping LQ Test(Qiagen).

Techniques: Diagnostic Assay, Amplification, Nested PCR, Multiplex PCR, Hybridization, Microarray, Suspension, Multiplex Assay, Real-time Polymerase Chain Reaction, HPV Assay, Southern Blot, In Situ Hybridization, Neutralization

Journal: Cold Spring Harbor Perspectives in Biology

Article Title: Chromatin Remodeling in Mammary Gland Differentiation and Breast Tumorigenesis

doi: 10.1101/cshperspect.a004515

Figure Lengend Snippet: Summary of candidate gene and genome-wide techniques for DNA methylation analysis a

Article Snippet: Microarray-based approaches , DMH (differential methylation hybridization): MTA (methylation tissue array); MSO (methylation-specific oligonucleotide); HELP ( Hpa II tiny fragment enrichment by ligation-mediated PCR); AIMS (amplification of intermethylated sites); MSNP (methylation single nucleotide polymorphism chip-based method); MMASS (microarray-based methylation assessment of single sample); PMAD (promoter-associated methylated DNA amplification); MSDK (methylation-specific digital karyotyping) MIAMI (microarray-based integrated analysis of methylation); MCAM (methylated CpG island amplification and microarray); MeDIP-chip (methylated DNA immunoprecipitation on microarray); MIRA-chip (methylated-CpG island recovery assay on microarray); Mcr BC ; MethylScope ; Pharmacologic unmasking analysis ; and Infinium BeadArray.

Techniques: Genome Wide, DNA Methylation Assay, Cloning, Methylation Sequencing, Methylation, Combined Bisulfite Restriction Analysis Assay, Microarray, Hybridization, Ligation, Amplification, DNA Amplification, Methylated DNA Immunoprecipitation, Immunoprecipitation, Next-Generation Sequencing, Methylated DNA Immunoprecipitation Sequencing

Journal: Medical Journal of the Islamic Republic of Iran

Article Title: Human papilloma virus: A review study of epidemiology, carcinogenesis, diagnostic methods, and treatment of all HPV-related cancers

doi: 10.47176/mjiri.35.65

Figure Lengend Snippet: The classification of commercially available diagnostic techniques for the detection and genotyping of HPV

Article Snippet: , , PCR-based fluorescent bead array , ❖ Microarray-based HPV genotyping assays ○ The PapilloCheck HPV Screening Test(greiner bio), ○ Clart HPV 2 – papillomavirus clinical arrays( Genomica), ○ HPV GenoArray Test Kit ( Hybribio), ○ GeneTrack HPV DNA Chip( Daejeon), ○ GeneSQUARE HPV Microarray,( Kurabo) ○ INFINITI HPV-HR QUAD (AutoGenomics), ○ PANArray HPV Genotyping Chip( Panagene), ○ GG HPVCHIP ( Goodgene) ❖ Suspension array based HPV genotyping assays ○ The Multiplex HPV Genotyping Kit (Multimetrix, Heidelberg) ○ digene HPV Genotyping LQ Test(Qiagen).

Techniques: Diagnostic Assay, Amplification, Nested PCR, Multiplex PCR, Hybridization, Microarray, Suspension, Multiplex Assay, Real-time Polymerase Chain Reaction, HPV Assay, Southern Blot, In Situ Hybridization, Neutralization