Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Hydroxylase inhibition abrogates TNF-alpha-induced intestinal epithelial damage by hypoxia-inducible factor-1-dependent repression of FADD.

doi: 10.4049/jimmunol.1002541

Figure Lengend Snippet: FIGURE 1. DMOG treatment stabilizes functionally active intestinal epithelial HIF-1a proteins in vitro and in vivo. Low basal HIF-1a protein levels are detectable by ELISA in IECs under normoxic conditions. A, An exposure for 24 h to DMOG (1 mM in DMEM) or 1% oxygen strongly augmented HIF-1a protein levels in HT29 cells in vitro. B, In vivo, 4 d of DMOG treatment (8 mg in 0.5 ml sterile PBS, i.p., every other day) resulted in stably enhanced HIF- 1a protein levels in freshly isolated terminal ileal enterocytes. C, In vitro, DMOG and 1% oxygen strongly induced transcription of VEGF. D, In vivo, DMOG treatment resulted in a 2-fold increase of VEGF protein in terminal ileal enterocytes. Each condition of in vitro experiments was tested 6-fold. In vivo results are of a single experiment with n = 3–5 mice per group. E, Immunohistochemistry for HIF-1a on terminal ileal sections (original mag- nification 3200) showed that DMOG stabilized HIF-1a protein mainly in the IECs. Transcriptional activity of DMOG-induced HIF-1a protein was verified by qRT-PCR (in vitro) and ELISA (in vivo) for VEGF, because its gene is typically HIF-1a–induced. pp , 0.05; ppp , 0.01; pppp , 0.001.

Article Snippet: Total inflammation score was calculated as the sum of A and B. HIF-1a and FADD small interfering RNA HT29 cells (human colon carcinoma epithelial cell line, HTB-38; American Type Culture Collection, Manassas, VA) were seeded at 150,000 cells per well in a 24-well plate and transfected the next day either with small interfering RNA (siRNA) duplexes targeting human HIF-1a or FADD or with control siRNA (Biolegio, Nijmegen, The Netherlands), using 5 ml lipofectamine (Invitrogen, Carlsbad, CA) in 500 ml RPMI 1640 medium (21, 22).

Techniques: In Vitro, In Vivo, Enzyme-linked Immunosorbent Assay, Sterility, Stable Transfection, Isolation, Immunohistochemistry, Activity Assay, Quantitative RT-PCR

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Hydroxylase inhibition abrogates TNF-alpha-induced intestinal epithelial damage by hypoxia-inducible factor-1-dependent repression of FADD.

doi: 10.4049/jimmunol.1002541

Figure Lengend Snippet: FIGURE 4. DMOG protects IECs against TNF-a–induced apoptosis in an HIF-1a–dependent manner. A, B, DMOG pretreatment (1 mM in DMEM for 24 h) prevented intestinal epithelial cells against TNF-a–induced apoptosis in vitro. Transfection of HT29 cells with HIF-1a-siRNA prior to DMOG treatment resulted in highly reduced HIF-1a protein stabilization (A) and significantly diminished protective effects of DMOG on TNF-a–induced IEC apoptosis (B). C, Four days of pretreatment with DMOG (8 mg in 0.5 ml sterile PBS, i.p., every other day) protected C57BL/6J mice against TNF-a– induced apoptosis in vivo. As such, DMOG-pretreated mice did not show the characteristic villous deformation due to the massive shedding of apoptotic enterocytes seen in vehicle-treated littermates after a single i.v. injection with rTNF-a (10 mg in 200 ml saline). Representative pictures of the TUNEL- stained section of the terminal ileum (original magnification 3200, detail magnification 3400) are given. Apoptotic cells have brown-stained nuclei (black arrows). Each condition of in vitro experiments was tested at least three times. In vivo results are of a single experiment with n = 3–5 mice per group. pp , 0.05; ppp , 0.01; pppp , 0.001.

Article Snippet: Total inflammation score was calculated as the sum of A and B. HIF-1a and FADD small interfering RNA HT29 cells (human colon carcinoma epithelial cell line, HTB-38; American Type Culture Collection, Manassas, VA) were seeded at 150,000 cells per well in a 24-well plate and transfected the next day either with small interfering RNA (siRNA) duplexes targeting human HIF-1a or FADD or with control siRNA (Biolegio, Nijmegen, The Netherlands), using 5 ml lipofectamine (Invitrogen, Carlsbad, CA) in 500 ml RPMI 1640 medium (21, 22).

Techniques: In Vitro, Transfection, Sterility, In Vivo, Injection, Saline, TUNEL Assay, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Hydroxylase inhibition abrogates TNF-alpha-induced intestinal epithelial damage by hypoxia-inducible factor-1-dependent repression of FADD.

doi: 10.4049/jimmunol.1002541

Figure Lengend Snippet: FIGURE 5. DMOG represses intestinal epithelial FADD, which renders IECs less susceptible to TNF-a-mediated apoptosis. A, Transcription of FADD was reduced 2.5- to 3-fold in HT29 IECs by a 24-h exposure to DMOG (1 mM in DMEM) or 1% oxygen. B, Reduction of HIF-1a protein by transfecting HT29 cells with HIF-1a siRNA prior to DMOG treatment resulted in significantly increased FADD transcription, confirming a significant role for HIF-1a in FADD inhibition. C, In vivo, a Western blot revealed strongly reduced FADD protein levels in the terminal ileal mucosa of C57BL/6J mice after 4 d of DMOG treatment (8 mg in 0.5 ml sterile PBS, i.p., every other day). D, FADD siRNA was used to check the functional consequences of FADD down- regulation on TNF-a-mediated apoptosis. E, siRNA repressed FADD 2.5- to 3-fold (similar to DMOG) in HT29 cells, resulting in undetectable protein levels by Western blot. F, FADD repression protected IECs against TNF-a-mediated apoptosis. Each condition of in vitro experiments was tested six times. In vivo results are representative of two independent experiments with n = 3–5 mice per group. pp , 0.05; ppp , 0.01; pppp , 0.001.

Article Snippet: Total inflammation score was calculated as the sum of A and B. HIF-1a and FADD small interfering RNA HT29 cells (human colon carcinoma epithelial cell line, HTB-38; American Type Culture Collection, Manassas, VA) were seeded at 150,000 cells per well in a 24-well plate and transfected the next day either with small interfering RNA (siRNA) duplexes targeting human HIF-1a or FADD or with control siRNA (Biolegio, Nijmegen, The Netherlands), using 5 ml lipofectamine (Invitrogen, Carlsbad, CA) in 500 ml RPMI 1640 medium (21, 22).

Techniques: Inhibition, In Vivo, Western Blot, Sterility, Functional Assay, In Vitro

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Hydroxylase inhibition abrogates TNF-alpha-induced intestinal epithelial damage by hypoxia-inducible factor-1-dependent repression of FADD.

doi: 10.4049/jimmunol.1002541

Figure Lengend Snippet: FIGURE 7. CD Patients with a normal appearing terminal ileum have increased FADD mRNA levels compared with those of healthy controls. A, Biopsy specimens of normal appearing terminal ileum of patients with CD and healthy controls were analyzed for mucosal FADD expression. Terminal ileal FADD transcription was enhanced significantly in CD patients versus healthy controls. B, Subanalysis showed that this finding was only true for patients with a history of ileal CD and not for CD patients that had never had ileal involvement. The qRT-PCR results were confirmed at the protein level by immunohis- tochemistry. Representative images (FADD, original magnification 3100, detail magnification 3200) are given. pp , 0.05; ppp , 0.01; pppp , 0.001.

Article Snippet: Total inflammation score was calculated as the sum of A and B. HIF-1a and FADD small interfering RNA HT29 cells (human colon carcinoma epithelial cell line, HTB-38; American Type Culture Collection, Manassas, VA) were seeded at 150,000 cells per well in a 24-well plate and transfected the next day either with small interfering RNA (siRNA) duplexes targeting human HIF-1a or FADD or with control siRNA (Biolegio, Nijmegen, The Netherlands), using 5 ml lipofectamine (Invitrogen, Carlsbad, CA) in 500 ml RPMI 1640 medium (21, 22).

Techniques: Expressing, Quantitative RT-PCR