Journal: Marine Drugs

Article Title: Anti-Inflammatory Effects of Marine-Derived Resorcylic Acid Lactone Derivatives in Ulcerative Colitis via the MAPK/ERK Pathway

doi: 10.3390/md24020069

Figure Lengend Snippet: Compound 4 exhibits potent in vivo anti-inflammatory activity in DSS-induced colitis. ( A ) Schematic diagram of colitis. C57BL/6 mice were given 3% DSS water for 8 days. 5-ASA (100 mg/kg) and compound 4 (10, 20 mg/kg) were administered daily by gavage for 9 consecutive days, and the mice were sacrificed on day 9. ( B ) At the end of the experiment, the representative results of colon length. The weight ( C ) and DAI score ( D ) of mice in each group were recorded daily. ( E ) spleen index of each group. ( F ) Histopathological images of the colon in each group of mice. Scale bar = 100 µm. ( G ) Expression levels of ZO-1, Occludin, and Claudin-1 in colon tissues of each group. ( H – K ) The concentration of LPS, TNF- α , IL-6, and IL-1 β in the serum of each group. The samples were derived from the same experiment, and the blots were processed in parallel. Data represent mean ± SD, ns: not significant; ## p < 0.01 vs. control group; ** p < 0.01 vs. DSS group.

Article Snippet: After blocking with 5% BSA for 2 h, the membranes were incubated at 4 °C overnight with primary antibodies against the following target proteins: β -Actin (CST, #4970), P-ERK (CST, #5726), ERK (CST, #4695), ZO-1 (Affinity, #AF5145), Occludin (Proteintech, #27260-1-AP), and Claudin-1 (Proteintech, #28674-1-AP).

Techniques: In Vivo, Activity Assay, Expressing, Concentration Assay, Derivative Assay, Control

Journal: Cancer Research

Article Title: Epigenetic Silencing of Occludin Promotes Tumorigenic and Metastatic Properties of Cancer Cells via Modulations of Unique Sets of Apoptosis-Associated Genes

doi: 10.1158/0008-5472.can-06-1864

Figure Lengend Snippet: Figure 1. Forced expression of occludin promotes apoptosis. A, cell death of HeLa-Vec and HeLa-Oc522aa cells was examined by two different assays (left, diphenylamine method used in this study; right, TUNEL assay) after 24 hours in the presence or absence of various concentrations of H2O2 with or without 1 Amol/L atRA treatment. B, siRNA targeted to occludin abrogated the apoptotic sensitization effects of occludin with or without 1 Amol/L atRA treatment in the presence of 100 Amol/L H2O2 (right). Western blot analysis confirmed the silencing effects of siRNA (left). C, constructs of deletion mutants to identify the gene locus responsible for the apoptotic sensitization effect (left). Membrane localizations were determined by distribution of enhanced green fluorescent protein (EGFP) signals under fluorescent microscopy after transient transfection with each deletion mutant linked to EGFP. Cell death was examined with a number of apoptotic inducers in HeLa cells transfected with various deletion mutants (right). D, effects of three types of kinase inhibitors on apoptosis. The cells were treated with PD98059, SB203580, or LY294002 for 24 hours before the apoptotic stimulation, and apoptosis was stimulated with 100 Amol/L H2O2 for 24 hours (left). Western blot analysis of pMAPK and ERK1/2 treated for 24 hours with (+) or without () 1 Amol/L atRA (right) in HeLa-Vec (Vec) and HeLa-Oc522aa (Oc) cells. The signals of pMAPK (p44) are normalized to the expression of ERK1/2 (p44) in each right panel and are defined as 100% in HeLa-Vec cells without the treatment. *, P < 0.05 versus control cells.

Article Snippet: Small interfering RNA (siRNA) targeted to occludin (Santa Cruz Biotechnologies, Santa Cruz, CA) was also transfected into the cells using FuGENE 6 reagent (Roche).

Techniques: Expressing, TUNEL Assay, Western Blot, Construct, Membrane, Microscopy, Transfection, Mutagenesis, Control

Journal: Cancer Research

Article Title: Epigenetic Silencing of Occludin Promotes Tumorigenic and Metastatic Properties of Cancer Cells via Modulations of Unique Sets of Apoptosis-Associated Genes

doi: 10.1158/0008-5472.can-06-1864

Figure Lengend Snippet: Figure 2. Occludin expression suppresses the tumorigenicity in vitro. A and B, anoikis was induced after adding cells to agarose-coated dishes to avoid cell attachment. DNA laddering analysis to confirm apoptotic cell death (A, left). Quantification of anoikis in the presence or absence of 1 Amol/L atRA as an apoptotic sensitizer in wt occludin (Oc522aa)–expressing various cancer cells (A, right), and siRNA effect in HeLa cells (B). siRNA (100 nmol/L) was transfected 48 hours before anoikis induction. C and D, occludin expression suppresses colony formation in two-dimensional (C) or three- dimensional (D) cultures of indicated cells. Quantifications of colonies in occludin522aa (Oc)–expressing cells and representative result of this assay (D, left). The number of colonies formed from control cells was defined as 100%. Insets, Ki67 labeling indexes in B16F10 cells. Bar, 250 Am. *, P < 0.05 versus control cells.

Article Snippet: Small interfering RNA (siRNA) targeted to occludin (Santa Cruz Biotechnologies, Santa Cruz, CA) was also transfected into the cells using FuGENE 6 reagent (Roche).

Techniques: Expressing, In Vitro, Cell Attachment Assay, DNA Laddering, Transfection, Control, Labeling

Journal: Cancer Research

Article Title: Epigenetic Silencing of Occludin Promotes Tumorigenic and Metastatic Properties of Cancer Cells via Modulations of Unique Sets of Apoptosis-Associated Genes

doi: 10.1158/0008-5472.can-06-1864

Figure Lengend Snippet: Figure 3. RARa agonists preferentially induce endogenous occludin. A, endogenous occludin expression is preferentially induced by RARa stimulation such as 1 Amol/L atRA, 100 nmol/L Am80, or 100 nmol/L Am580 for 24 hours. Quantification of expression levels is shown below the gel image. B, cells were pretreated with (+) or without () 1 Amol/L atRA 24 hours before apoptotic stimulation. The cell deaths were quantitated after 24 hours in the presence of 100 Amol/L H2O2 in combination with (+) or without () 100 nmol/L occludin-specific siRNA transfection. *, P < 0.05 versus cells without siRNA transfection. C, colony forming efficacy was evaluated after 24 hours treatment with 1 Amol/L atRA, 100 nmol/L Am80, or 100 nmol/L Am580 in two-dimensional (left) and three-dimensional (right) cultures. The cells pretreated with DMSO (vehicle) were used as a control and defined as 100% for colony forming efficacy.

Article Snippet: Small interfering RNA (siRNA) targeted to occludin (Santa Cruz Biotechnologies, Santa Cruz, CA) was also transfected into the cells using FuGENE 6 reagent (Roche).

Techniques: Expressing, Transfection, Control

Journal: Cancer Research

Article Title: Epigenetic Silencing of Occludin Promotes Tumorigenic and Metastatic Properties of Cancer Cells via Modulations of Unique Sets of Apoptosis-Associated Genes

doi: 10.1158/0008-5472.can-06-1864

Figure Lengend Snippet: Figure 5. Occludin expression suppresses tumorigenicity and lung metastasis in vivo. A and B, B16F10 and AC2M2 cells transfected with occludin522aa (Oc) grow more slowly than controls (Vec) in allograft transplantation models. A, representative developed tumors and their weights. Tumor volume was calculated in two dimensions in a time-dependent manner after inoculation of the vector (square symbol and solid line) or occludin522aa-transfected (triangle symbol and dotted line) cells in the first (filled) and second (open) lines of experiments (B). Bar, 1 cm. C, quantitative analysis of lung metastasis in mice following transplantation of B16F10 and AC2M2 cells transfected with vector (Vec) and wt occludin [Oc522aa; B16F10 (2 104 cells per animal): Vec, n = 8; Oc, n = 10; AC2M2 (2.5 104 cells per animal): Vec, n = 16; Oc, n = 15; AC2M2 (7.5 103 cells per animal): Vec, n = 8; Oc, n = 12]. Mice were sacrificed after 21 days in B16F10 cells, 34 F 2.1 days in AC2M2-Vec, and 37 F 2.8 days in AC2M2-Oc522aa cells postinoculation. *, P < 0.05 versus control group.

Article Snippet: Small interfering RNA (siRNA) targeted to occludin (Santa Cruz Biotechnologies, Santa Cruz, CA) was also transfected into the cells using FuGENE 6 reagent (Roche).

Techniques: Expressing, In Vivo, Transfection, Transplantation Assay, Plasmid Preparation, Control

Journal: Cancer Research

Article Title: Epigenetic Silencing of Occludin Promotes Tumorigenic and Metastatic Properties of Cancer Cells via Modulations of Unique Sets of Apoptosis-Associated Genes

doi: 10.1158/0008-5472.can-06-1864

Figure Lengend Snippet: Figure 6. Occludin expression is epigenetically silenced by CpG island promoter methylation and affects multifaceted signaling pathways. A and B, MSP analysis to detect the unmethylated (U) and methylated (M) alleles on CpG islands of the occludin promoter (A, top). Synergy with (+) or without () 1 Amol/L 5¶-Aza-dC and 100 nmol/L trichostatin A (TSA) induces endogenous occludin (A, bottom), which is sufficient for apoptotic sensitization in the presence (+) or absence () of 100 nmol/L occludin-specific siRNA (B). C, colony-forming efficacy after either 5¶-Aza-dC (1 Amol/L) alone or 5¶-Aza-dC (1 Amol/L) and trichostatin A (100 nmol/L) treatment in two-dimensional (left) and three- dimensional (right) cultures. Occludin522aa- overexpressing cells (Cell-Oc522aa) were used as a control. D, gene expression profile in occludin-overexpressing HeLa cells in an apoptosis pathway–specific array. We identified a number of genes that increased or decreased by an average change of z100% or V100% (n = 2), which are shown in the graph in functional gene groupings.

Article Snippet: Small interfering RNA (siRNA) targeted to occludin (Santa Cruz Biotechnologies, Santa Cruz, CA) was also transfected into the cells using FuGENE 6 reagent (Roche).

Techniques: Expressing, Methylation, Protein-Protein interactions, Control, Gene Expression, Functional Assay

Journal: Journal of Clinical Biochemistry and Nutrition

Article Title: Tocotrienol improves learning and memory deficit of aged rats

doi: 10.3164/jcbn.15-52

Figure Lengend Snippet: Effect of T-3 on the disruption of BBB component proteins. (a) The expression of claudin-5 in aged and young rats. * p <0.02 vs young control. (b) The expression of occludin in aged and young rats. * p <0.03 vs young control. (c) The expression of JAM in aged and young rats. * p <0.03 vs young control; n = 9 for each group.

Article Snippet: Antibodies: Anti-c-Src (B-12), p-ERK1/2 (Thr 202/Tyr 204) and junctional adhesion molecule-A (JAM-A) (H-80) were purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany); anti-occludin was from ProSci Incorporated (Poway, CA); anti-claudin-5 (S201) pAb was from Bioworld Technology, Inc. (Louis Park, MN).

Techniques: Expressing