Journal: Journal of Cellular and Molecular Medicine

Article Title: A complex intronic enhancer regulates expression of the CFTR gene by direct interaction with the promoter

doi: 10.1111/j.1582-4934.2008.00621.x

Figure Lengend Snippet: DNase-chip detects cell-type-specific DHS 10 kb into the first intron of CFTR . ( A ) Shown are DHS tracks in four different cell types: CFTR - primary human skin fibroblasts, CFTR + human bronchial epithelial cell line 16HBE14o- and CFTR + human colon carcinoma cell lines Caco2 and HT29. Significant peaks representing DHS are apparent in the 5′ promoter region of CFTR + cells, whereas Caco2 and HT29 cells display another significant DHS within the first intron. Peak height is a measurement of –log10 ( P -value) between 0 and 16, as determined by ACME (see Methods). The sequence associated with the identified intronic DHS is shown in panel B . Highlighted sequence corresponds to previously identified positive regulatory element 7/8 . Sequence is numbered based on GenBank reference sequence AC000111. (Sequence corresponds to bases 116916908–116918007 of human genome build 17.) ( C ) CFTR expression in cell types used in this study measured by RT-PCR analysis compared with a β-glucocerebrosidase housekeeping gene control.

Article Snippet: Primary skin fibroblasts (ATCC: GM08333) were grown in MEM (Gibco) supplemented with 15% FBS.

Techniques: Sequencing, Expressing, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: A complex intronic enhancer regulates expression of the CFTR gene by direct interaction with the promoter

doi: 10.1111/j.1582-4934.2008.00621.x

Figure Lengend Snippet: The 7/8 enhancer directly interacts with the CFTR promoter. Chromosome conformation analysis of CFTR + Caco2 ( A ), CFTR + 16HBE14o- ( B ) and CFTR - primary skin fibroblasts ( C ) show higher interaction of the first intron with the promoter in cells with the intron 1 DHS. Cross-linked chro-matin from each cell type was digested with Hind III (sites indicated by dashed lines) and re-ligated. The interaction frequency between a fixed Hind III fragment at the CFTR promoter and Hind III fragments 5′ and within the first, third and seventh introns was measured by Taqman® quantitative PCR. Each reaction was normalized to a control region in the ERCC3gene, as previously described . The amplification efficiencies of all primer sets were verified using Hind III-digested CFTR and ERCC3 BACs. Each experiment was repeated at least twice; a single representative experiment is shown with PCR reactions performed in triplicate. Error bars represent standard error of the mean ( n = 3).

Article Snippet: Primary skin fibroblasts (ATCC: GM08333) were grown in MEM (Gibco) supplemented with 15% FBS.

Techniques: Real-time Polymerase Chain Reaction, Control, Amplification

Journal: Arthritis Research & Therapy

Article Title: Differential effects of Th1 versus Th2 cytokines in combination with hypoxia on HIFs and angiogenesis in RA

doi: 10.1186/ar3934

Figure Lengend Snippet: Angiogenesis RT² Profiler™ PCR Array data and expression analysis of human rheumatoid arthritis cells . PCR arrays were used with cDNA from rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) from five RA patients and incubated for either (a) 4 hours or (b) 24 hours in 1% oxygen (hypoxia) and (c) from three RA patients exposed to 1 mM dimethyloxalylglycine (DMOG) for 24 hours. Values are fold-change versus normoxia (21% oxygen) or dimethyl sulfoxide (DMSO) control and were analysed against housekeeping gene β-actin, 18S ribosomal RNA, hypoxanthine phosphoribosyltransferase 1 and 60S ribosomal protein L13a. Genes significantly increased or decreased by a factor ≥2 ( P < 0.05, dotted line) in all patients are shown. Data are mean ± standard error of the mean and were analysed by paired t test of ΔCt values, comparing normoxia versus hypoxia (a and b) or DMSO control versus DMOG (c). (d) Western blotting demonstrates induction of hypoxia-inducible factor (HIF)-1α and HIF-2α in total protein lysates from RA FLS in response to 1% oxygen or 1 mM DMOG for 24 hours. An antibody against α-tubulin was used as a loading control. A lane irrelevant to the study was removed as indicated by the line to show lanes of interest adjacent to one another. (e) Freshly dissociated total human RA synovial membrane cells from four patients were analysed for expression of vascular endothelial growth factor (VEGF), leptin, ephrin A3 (EFNA3) and angiopoietin-like (ANGPTL)-4 using quantitative PCR. Changes in mRNA are expressed as fold-change relative to levels under 21% oxygen set as 1.0 (dotted line). * P < 0.05, ** P < 0.01, *** P < 0.001. EFNB2, ephrin B2; FIGF, C-fos-induced growth factor (VEGF D); HAND2, Heart and neural crest derivatives expressed 2; HPSE, heparanase; ID3, inhibitor of DNA-binding 3, dominant negative helix-loop-helix protein; NRP2, neuropillin 2; PLAU, plasminogen activator urokinase.

Article Snippet: FLS and normal human skin fibroblasts (HSF; Lonza, Walkersville, MD, USA) were cultured in DMEM containing 10% foetal bovine serum, 4.5 g/l glucose and L -glutamine, supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (PAA Laboratories, Coelbe, Germany).

Techniques: Expressing, Incubation, Control, Western Blot, Membrane, Real-time Polymerase Chain Reaction, Binding Assay, Dominant Negative Mutation

Journal: Arthritis Research & Therapy

Article Title: Differential effects of Th1 versus Th2 cytokines in combination with hypoxia on HIFs and angiogenesis in RA

doi: 10.1186/ar3934

Figure Lengend Snippet: Hypoxia-induced hypoxia-inducible factor isoform dependence of angiogenic genes in human rheumatoid arthritis fibroblast-like synoviocytes . Hypoxia-induced hypoxia-inducible factor (HIF) isoform dependence of ephrin A3 (EFNA3), angiopoietin-like (ANGPTL)-4, leptin and vascular endothelial growth factor (VEGF) in human rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). RA FLS were transiently transfected with siRNA oligonucleotides complementary to hypoxia-inducible factor (HIF)-1α (siHIF-1α) or HIF-2α (siHIF-2α) or both simultaneously. An siRNA oligonucleotide complementary to siLuc was used as control. The cell cultures were subsequently exposed to 1% oxygen (hypoxia) for 24 hours. Total RNA was isolated and cDNA generated, and the mRNA level of (a) leptin and (c) ANGPTL-4, (e) VEGF and (g) EFNA3 was determined using quantitative PCR. Changes in mRNA expressed as fold-change relative to levels in the siLuc transfected normoxic controls set as 1.0 (dotted line). The secretion of (b) leptin, (d) ANGPTL-4 and (f) VEGF protein was measured using ELISA. Data expressed as mean ± standard error of the mean of ≥3 independent experiments with sample assayed in triplicate, and were analysed using one-way analysis of variance with Bonferroni's post-hoc test for multiple comparisons versus hypoxic siLuc transfected cells (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: FLS and normal human skin fibroblasts (HSF; Lonza, Walkersville, MD, USA) were cultured in DMEM containing 10% foetal bovine serum, 4.5 g/l glucose and L -glutamine, supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (PAA Laboratories, Coelbe, Germany).

Techniques: Transfection, Control, Isolation, Generated, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Arthritis Research & Therapy

Article Title: Differential effects of Th1 versus Th2 cytokines in combination with hypoxia on HIFs and angiogenesis in RA

doi: 10.1186/ar3934

Figure Lengend Snippet: Proangiogenic/anti-angiogenic effects of Th1 and Th2 cytokines on rheumatoid arthritis fibroblast-like synoviocyte gene expression . Cell cultures were exposed to 1% oxygen (hypoxia) and/or 10 ng/ml cytokine or left untreated for 24 hours. Total RNA was isolated and cDNA generated, and the mRNA level of (a) vascular endothelial growth factor (VEGF), (c) angiopoietin-like (ANGPTL)-4, (e) leptin and (f) ephrin A3 (EFNA3) was determined using quantitative PCR. Changes in mRNA expressed as fold-change relative to levels in untreated samples set as 1.0 (dotted line). Secretion of (b) VEGF and (d) ANGPTL-4 protein was measured using ELISA. Data expressed as the mean ± standard error of the mean of ≥3 independent experiments with samples assayed in triplicate. Samples analysed using one-way analysis of variance with Bonferroni's post-hoc test for multiple comparisons versus control normoxia († P < 0.05, †† P < 0.01, ††† P < 0.001) or versus hypoxia alone (* P < 0.05, ** P < 0.01,*** P < 0.001).

Article Snippet: FLS and normal human skin fibroblasts (HSF; Lonza, Walkersville, MD, USA) were cultured in DMEM containing 10% foetal bovine serum, 4.5 g/l glucose and L -glutamine, supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (PAA Laboratories, Coelbe, Germany).

Techniques: Gene Expression, Isolation, Generated, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Control

Journal: Arthritis Research & Therapy

Article Title: Differential effects of Th1 versus Th2 cytokines in combination with hypoxia on HIFs and angiogenesis in RA

doi: 10.1186/ar3934

Figure Lengend Snippet: Th1-induced vascular endothelial growth factor but not angiopoietin-like-4 expression is dependent on hypoxia-inducible factor-1 . Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) were transiently transfected with siRNA oligonucleotides complementary to hypoxia-inducible factor (HIF)-1α (siHIF-1α). An siRNA oligonucleotide complementary to siLuc was used as control. The cell cultures were subsequently exposed to 10 ng/ml cytokines or left untreated for 24 hours. Changes in mRNA in response to IL-1β or TNFα of (a) vascular endothelial growth factor (VEGF) and (b) angiopoietin-like (ANGPTL)-4 expressed as fold-change relative to levels in the siLuc transfected untreated controls set as 1.0 (dotted line). Data expressed as the mean ± standard error of the mean of ≥3 independent experiments with samples assayed in triplicate and analysed versus cytokine-treated siLuc transfected cells. * P < 0.05, ** P < 0.01.

Article Snippet: FLS and normal human skin fibroblasts (HSF; Lonza, Walkersville, MD, USA) were cultured in DMEM containing 10% foetal bovine serum, 4.5 g/l glucose and L -glutamine, supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (PAA Laboratories, Coelbe, Germany).

Techniques: Expressing, Transfection, Control

Journal: Arthritis Research & Therapy

Article Title: Differential effects of Th1 versus Th2 cytokines in combination with hypoxia on HIFs and angiogenesis in RA

doi: 10.1186/ar3934

Figure Lengend Snippet: Interactions of hypoxia and cytokines modulate induction of angiogenic activity by rheumatoid arthritis fibroblast-like synoviocytes . Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) were cultured in normoxia (21% oxygen), hypoxia (1% oxygen) or with 10 ng/ml IL-4 or TNFα for 24 hours. Alternatively the cells were co-stimulated with cytokines whilst cultured in hypoxia to mimic the environment in RA joints. Supernatants from these cultures were applied to wells of a 96-well plate containing growth factor-reduced matrigel and pre-seeded human microvascular endothelial cell (HMEC)-1 cells and left to form tubules for 4 to 6 hours. (a) A single picture was captured from the centre of each well with a camera (QICAM FAST; QImaging) attached to a microscope (CKX41; Olympus), with a representative example for each condition shown here. Using AngioSys Image Analysis software we analysed several parameters of angiogenesis: (b) percentage of field area covered by HMEC-1 tubules, (c) number of tubules, and (d) tubule junctions formed in the field area. Data expressed as the mean ± standard error of the mean of ≥3 independent experiments with supernatants from each condition assayed in triplicate wells. Samples analysed using one-way analysis of variance with Bonferroni's post-hoc test for multiple comparisons versus control (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: FLS and normal human skin fibroblasts (HSF; Lonza, Walkersville, MD, USA) were cultured in DMEM containing 10% foetal bovine serum, 4.5 g/l glucose and L -glutamine, supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (PAA Laboratories, Coelbe, Germany).

Techniques: Activity Assay, Cell Culture, Microscopy, Software, Control