Journal: The Journal of Biological Chemistry

Article Title: ETS-related Gene (ERG) Controls Endothelial Cell Permeability via Transcriptional Regulation of the Claudin 5 (CLDN5) Gene *

doi: 10.1074/jbc.M111.300236

Figure Lengend Snippet: ERG knockdown increases EC permeability. HUVEC were infected with lentiviral ERG shRNA (ERG-sh) or a CTR shRNA (CTR-sh). A and B, cell extracts were prepared, and equal amounts of total proteins were separated on polyacrylamide gel. Protein levels of ERG and the control protein, tubulin, were determined by Western blot analysis. A, representative Western blot (IB) shows protein bands of ERG (top) and tubulin (bottom). B, densitometry analysis shows density ratios of ERG and tubulin. Three independent experiments were performed. *, p < 0.01. C, in FITC-dextran Transwell assay, ERG shRNA-treated or control shRNA-treated HUVEC were seeded onto collagen-coated inserts. When the EC monolayer became confluent, FITC-dextran was added to the top. Permeability was determining by measuring the fluorescence within the lower chamber after a 6-h incubation. The data are presented compared with control cells ± S.D. (error bars), n = 5. p < 0.01. D, in ECIS assay, the intercellular resistance was measured 24 h after cells were plated. The data represent the relative barrier resistance (Rb) values relative to control cells at the indicated time points, ± S.D., n = 3. *, p < 0.01. E, effects of ERG knockdown on stress fiber and gap formation are shown. Cells were fixed and stained with phalloidin-594 (red) to demonstrate cytoskeletal actin in either ERG or control (CTR) shRNA transfected HUVEC. Arrows indicate intercellular gaps.

Article Snippet: Briefly, 8-well ECIS arrays (8W10E+) were coated with fibronectin (Invitrogen).

Techniques: Permeability, Infection, shRNA, Western Blot, Transwell Assay, Fluorescence, Incubation, Staining, Transfection

Journal: The Journal of Biological Chemistry

Article Title: ETS-related Gene (ERG) Controls Endothelial Cell Permeability via Transcriptional Regulation of the Claudin 5 (CLDN5) Gene *

doi: 10.1074/jbc.M111.300236

Figure Lengend Snippet: Partial rescue of TNF-α-induced decreased EC intercellular resistance by ERG. A, HUVEC infected with adenoviruses expressing ERG or GFP control (CTR). Overexpression of ERG was determined by Western blotting (IB) using antibody against ERG. Tubulin was used as a loading control. B, ECIS assay. HUVEC infected with Ad-ERG or Ad-CTR were incubated in medium containing TNF-α (2 ng/ml) or medium alone for 24 h. The intercellular resistance was measured and collected using ECIS system over 24 h. The data are shown relative to control cells in the absence of TNF-α stimulation. The bar graphs represent the relative Rb values at the indicated time points, ± S.D. (error bars), n = 3. *, p < 0.01.

Article Snippet: Briefly, 8-well ECIS arrays (8W10E+) were coated with fibronectin (Invitrogen).

Techniques: Infection, Expressing, Over Expression, Western Blot, Incubation

Journal: The Journal of Biological Chemistry

Article Title: ETS-related Gene (ERG) Controls Endothelial Cell Permeability via Transcriptional Regulation of the Claudin 5 (CLDN5) Gene *

doi: 10.1074/jbc.M111.300236

Figure Lengend Snippet: CLDN5 knockdown is associated with decreased intercellular resistance. A and B, ability of CLDN5 siRNAs to repress CLDN5 expression. HUVEC were transfected with various CLDN5 siRNAs (#1–#4; 40 nmol/liter) or a control siRNA (CTR). After incubation for 48 h, CLDN5 expression was assessed by QPCR (A) using CLDN5-specific primers or by Western blot analysis (B) using anti-CLDN5 antibody. Tubulin was used as a loading control. Quantitation was done by densitometric analysis. n = 3. *, p < 0.05. C, ECIS. EC were treated with CLDN5 #2, #4 siRNAs or a control siRNA (40 nmol/liter). After incubation for 48 h cells were then plated on the 8W10E+ to assess the intercellular resistance for a period of 24 h. Data shown are presented as values of normalized Rb value relative to control samples. The bar graphs represent the relative Rb values at indicated time points relative to control ± S.D., n = 3. *, p < 0.01.

Article Snippet: Briefly, 8-well ECIS arrays (8W10E+) were coated with fibronectin (Invitrogen).

Techniques: Expressing, Transfection, Incubation, Western Blot, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: ETS-related Gene (ERG) Controls Endothelial Cell Permeability via Transcriptional Regulation of the Claudin 5 (CLDN5) Gene *

doi: 10.1074/jbc.M111.300236

Figure Lengend Snippet: Adenoviral delivery of CLDN5 partially rescues the effect of ERG knockdown on intercellular resistance. A, HUVEC treated with ERG shRNA and infected with either Ad-CLDN5 or Ad-GFP as a control. After incubation for 48 h, the overexpression of CLDN5 was determined by Western blotting using antibody against CLDN5. Tubulin was used as a loading control. B, ECIS assay. HUVEC infected with ERG shRNA or control shRNA were cultured and transfected with either Ad-CLDN5 or Ad-GFP as a control. After incubation for 48 h the intercellular resistance was measured and collected using ECIS system for 24 h. The data are shown relative to control cells treated with control Ad-GFP (Ad-CTR). The bar graphs represent the relative Rb values at indicated time points relative to control ± S.D., n = 3. *, p < 0.01.

Article Snippet: Briefly, 8-well ECIS arrays (8W10E+) were coated with fibronectin (Invitrogen).

Techniques: shRNA, Infection, Incubation, Over Expression, Western Blot, Cell Culture, Transfection