Journal: International Journal of Molecular Sciences

Article Title: Rel Family Transcription Factor NFAT5 Upregulates COX2 via HIF-1α Activity in Ishikawa and HEC1a Cells

doi: 10.3390/ijms25073666

Figure Lengend Snippet: Effect of NFAT5 on COX2 transcript and protein levels in Ishikawa cells. ( a ). Ishikawa cells were treated with 0.5 mM DMOG for 24 h. mRNA expression level of NFAT5 was quantified by qRT-PCR. Data were normalized to L19 and presented as mean ± SEM. ( n = 6; *, p < 0.05). ( b ). NFAT5 protein abundance was investigated by SDS-PAGE and western blot analysis. Ishikawa cells were treated with 0.5 mM DMOG for 24 h. Data were normalized to each corresponding level of pan-actin and shown as mean ± SEM. ( n = 6; ****, p < 0.0001, a.u: arbitrary unit). ( c ). mRNA expression level of NFAT5 , HIF1A , and PTGS2 were quantified by qRT-PCR. Ishikawa cells were transfected with NFAT5 overexpression plasmid for 24 h ( n = 5; **, p < 0.01, ****, p < 0.0001). ( d ). NFAT5 and PTGS2 protein abundance were investigated by SDS-PAGE and western blot analysis using the indicated antibodies. Ishikawa cells were transfected with NFAT5 overexpression plasmid for 24 h ( n = 6; *, p < 0.05; **, p < 0.01). ( e ). Luciferase activity of HIF-1α that was normalized to renilla post Ishikawa cells transfected with NFAT5 overexpression plasmid for 24 h ( n = 6; ****, p < 0.0001). ( f ). mRNA expression level of PTGS2 was quantified by qRT-PCR. Ishikawa cells were treated with 0.5 mM DMOG for 24 h ( n = 6; **, p < 0.01). ( g ). COX2 protein abundance was investigated by SDS-PAGE and western blot analysis using the indicated antibodies. Ishikawa cells were treated with 0.5 mM DMOG for 24 h ( n = 6; **, p < 0.01).

Article Snippet: Ishikawa or HEC1a cells were seeded onto 24-well plates at a density of 5 × 10 4 cells/well with 10% FBS DMEM and allowed to attach for 24 h. Next, cells were transfected with HIF-1α vector (#87261, Addgene, Watertown, MA, USA) using Lipofectamine LTX with Plus reagent (#15338100, Invitrogen) as per the manufacturer’s instruction.

Techniques: Expressing, Quantitative RT-PCR, SDS Page, Western Blot, Transfection, Over Expression, Plasmid Preparation, Luciferase, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Rel Family Transcription Factor NFAT5 Upregulates COX2 via HIF-1α Activity in Ishikawa and HEC1a Cells

doi: 10.3390/ijms25073666

Figure Lengend Snippet: Synergistic effect of NFAT5 overexpression and DMOG on COX2 transcript and protein levels in Ishikawa cells. ( a ). mRNA expression level of NFAT5 and PTGS2 quantified by qRT-PCR. Ishikawa cells were treated with 0.5 mM DMOG for 24 h post with or without 24 h transfection with NFAT5 overexpression plasmid. Data were normalized to L19 and presented as mean ± SEM. (NFAT5, n = 6; *, p < 0.05; **, p < 0.01). ( b – d ). NFAT5 and COX2 protein abundance were investigated by SDS-PAGE and western blot analysis using the indicated antibodies. Ishikawa cells were subjected to with or without NFAT5 transfection followed by treatment with or without DMOG. Data were normalized to each corresponding level of pan-actin and shown as mean ± SEM. ( n = 5; *, p < 0.05; **, p < 0.01; ***, p < 0.001, ****, p < 0.0001, a.u: arbitrary unit). ( e ). Effect of hypoxia on HIF-1α induction with or without NFAT5 transfection using Luciferase promoter assay. Data shown as mean ± SEM. ( n = 6; **, p < 0.01). ( f ). Effect on PGE2 levels with or without NFAT5 transfection, followed by treatment with or without DMOG (0.5 mM, 24 h). Data shown as mean ± SEM. ( n = 4; *, p < 0.05).

Article Snippet: Ishikawa or HEC1a cells were seeded onto 24-well plates at a density of 5 × 10 4 cells/well with 10% FBS DMEM and allowed to attach for 24 h. Next, cells were transfected with HIF-1α vector (#87261, Addgene, Watertown, MA, USA) using Lipofectamine LTX with Plus reagent (#15338100, Invitrogen) as per the manufacturer’s instruction.

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, SDS Page, Western Blot, Luciferase, Promoter Assay

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Activation of AMP-Activated Protein Kinase α and Extracelluar Signal-Regulated Kinase Mediates CB-PIC-Induced Apoptosis in Hypoxic SW620 Colorectal Cancer Cells

doi: 10.1155/2013/974313

Figure Lengend Snippet: CB-PIC (0 or 40 μ g/mL) was treated under normoxic or hypoxic conditions for various times (0, 0.5, 1, 2, 4, or 6 hrs). Western blotting was performed to determine HIF-1 α , AMPK α , pAMPK α , pERK, ERK, and β -actin expressions. (a) CB-PIC dramatically induces hypoxia-induced pAMPK α and pERK expressions and decreases hypoxia inducible factor 1 α (HIF-1 α ) accumulation in SW620 cells under hypoxia as time-dependent manner. (b) CB-PIC induced pAMPK α , and pERK expression was increased.

Article Snippet: Anti-rabbit IgG heavy and light chain-specific (rabbit, mouse) peroxidase conjugates and antibody against HIF-1 α were purchased from Gentex (Irvine, CA, USA).

Techniques: Western Blot, Expressing

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Activation of AMP-Activated Protein Kinase α and Extracelluar Signal-Regulated Kinase Mediates CB-PIC-Induced Apoptosis in Hypoxic SW620 Colorectal Cancer Cells

doi: 10.1155/2013/974313

Figure Lengend Snippet: (a) SW620 cells were treated with CB-PIC (40 μ g/mL) and/or PD 98059(ERK inhibitor 5 μ M) for 2 hrs under hypoxia. Western blotting was performed to determine HIF-1 α , AMPK α , pAMPK α , pERK, ERK, and β -actin expressions. (b) AMPK α siRNA decreases the activity of hypoxia-induced apoptosis in SW620 cells under hypoxia. Cells were transiently transfected with AMPK α siRNA or control siRNA in the presence or absence of CB-PIC (40 μ g/mL) under hypoxia. Western blotting was performed to determine the expression of HIF-1 α , AMPK α , pAMPK α , pERK, ERK, and β -actin.

Article Snippet: Anti-rabbit IgG heavy and light chain-specific (rabbit, mouse) peroxidase conjugates and antibody against HIF-1 α were purchased from Gentex (Irvine, CA, USA).

Techniques: Western Blot, Activity Assay, Transfection, Expressing

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Activation of AMP-Activated Protein Kinase α and Extracelluar Signal-Regulated Kinase Mediates CB-PIC-Induced Apoptosis in Hypoxic SW620 Colorectal Cancer Cells

doi: 10.1155/2013/974313

Figure Lengend Snippet: (a) Several concentrations of metformin (0, 0.5, 1, and 5 mM) were treated under hypoxia for 2 hr to determine the expression of AMPK α , pAMPK α , and β -actin. (b) SW 620 cells were treated with CB-PIC (40 μ g/mL) and/or metformin (5 mM) for 2 hr under hypoxia to investigate of effect of AMPK activator with CB-PIC (40 μ g/mL). Western blotting was performed to determine HIF-1 α , AMPK α , pAMPK α , pERK, ERK, pmTOR, pAKT, AKT, pACC, and β -actin expressions.

Article Snippet: Anti-rabbit IgG heavy and light chain-specific (rabbit, mouse) peroxidase conjugates and antibody against HIF-1 α were purchased from Gentex (Irvine, CA, USA).

Techniques: Expressing, Western Blot

Journal: Thrombosis Research

Article Title: Upregulation of hypoxia-inducible factor 1 alpha in local vein wall is associated with enhanced venous thrombus resolution

doi: 10.1016/j.thromres.2011.05.006

Figure Lengend Snippet: HIF1α staining of thrombosed IVC. Nucleated cells within the (A) non-thrombosed and (B) thrombosed (T) IVC stained positively for HIF1α (black).

Article Snippet: Transverse paraffin-sections (5 μm) of 1 and 7 day old thrombosed IVC (n = 3) were immunostained for HIF1α (anti-HIF1α, Stratech, UK) following antigen retrieval by pressure cooking sections in citrate buffer (0.3% sodium citrate, pH 6).

Techniques: Staining

Journal: Thrombosis Research

Article Title: Upregulation of hypoxia-inducible factor 1 alpha in local vein wall is associated with enhanced venous thrombus resolution

doi: 10.1016/j.thromres.2011.05.006

Figure Lengend Snippet: HIF1α, VEGF, and PLGF expression in thrombosed IVC. (A) HIF1α expression did not change throughout thrombus resolution or when compared with the non-thrombosed (NT) IVC. (B) VEGF was elevated at days 1 and 3 but not days 7 and 14, compared with the non-thrombosed (NT) IVC and was greater at day 1 compared with days 3, 7, and 14. (C) PLGF was higher at days 1, 3, and 7 after thrombus induction compared with day 14 and the non-thrombosed (NT) IVC. *P < 0.01 vs. NT, day 7, and day 14. **P < 0.0001 vs. NT and P < 0.01 vs. day 14. ***P < 0.0001 vs. NT and P < 0.001 vs. days 3, 7, and 14.

Article Snippet: Transverse paraffin-sections (5 μm) of 1 and 7 day old thrombosed IVC (n = 3) were immunostained for HIF1α (anti-HIF1α, Stratech, UK) following antigen retrieval by pressure cooking sections in citrate buffer (0.3% sodium citrate, pH 6).

Techniques: Expressing

Journal: Thrombosis Research

Article Title: Upregulation of hypoxia-inducible factor 1 alpha in local vein wall is associated with enhanced venous thrombus resolution

doi: 10.1016/j.thromres.2011.05.006

Figure Lengend Snippet: The effect of L-mim on HIF1α and HIF1-mediated angiogenic factor expression in thrombosed IVC. Values are pg/mg.

Article Snippet: Transverse paraffin-sections (5 μm) of 1 and 7 day old thrombosed IVC (n = 3) were immunostained for HIF1α (anti-HIF1α, Stratech, UK) following antigen retrieval by pressure cooking sections in citrate buffer (0.3% sodium citrate, pH 6).

Techniques: Expressing, Control

Journal: Thrombosis Research

Article Title: Upregulation of hypoxia-inducible factor 1 alpha in local vein wall is associated with enhanced venous thrombus resolution

doi: 10.1016/j.thromres.2011.05.006

Figure Lengend Snippet: Associations between HIF1α and VEGF or PLGF expression in the thrombosed IVC of mice treated with L-mim. Positive correlations between HIF1α and (A) VEGF (n = 17, R = 0.75, P < 0.001) or (B) PLGF (n = 17, R = 0.65, P < 0.01).

Article Snippet: Transverse paraffin-sections (5 μm) of 1 and 7 day old thrombosed IVC (n = 3) were immunostained for HIF1α (anti-HIF1α, Stratech, UK) following antigen retrieval by pressure cooking sections in citrate buffer (0.3% sodium citrate, pH 6).

Techniques: Expressing