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Nübel et al, Fig. 1 - Carcinogenesis

Nübel et al, Fig. 1 - Carcinogenesis

B: EA.hy-926

B: EA.hy-926 endothelial cells were exposed to TNFα (1 ng/ml), IR (10 Gy), UV-C-light (40 J/m2) and doxorubicin (3 µM). 5h after exposure cells were harvested for determination of Eselectin expression by ELISA-based method as described. Data shown are mean values ± sd from three independent experiments each performed in triplicate. C: Subconfluent HUVEC were exposed to increasing concentration TNFα. Immediately afterwards, cells were irradiated or not with 2 Gy. After incubation period of 5 h, E-selectin expression was analyzed by ELISA. Data shown are the mean ± sd from two independent experiments each performed in triplicate. Fig. 5: Rho GTPases are involved in the regulation of E-selectin gene expression by IR. A: Human endothelial cells (HUVEC) were left untreated (Con) or were pretreated overnight with Rho-inactivating clostridial Toxin A (175ng/ml). After pretreatment period cells were exposed to γ-rays (2 Gy). Five hours after exposure, cells were harvested for analysis of Eselectin expression by ELISA-based method described in Methods. Data shown are mean values ± sd from at least three independent experiments each performed in triplicate. B: 24h after transfection of EA.hy-926 cells with E-selectin promoter luciferase construct, cells were pretreated or not (Con) with ToxA (175 ng/ml), which specifically inactivates small GTPases of the Rho family [39,40]. Thereafter, cells were stimulated with IR (10 Gy). After overnight incubation, cells were harvested for determination of luciferase activity. Data shown are mean values ± sd from at least three independent experiments each performed in triplicate. Identical results were obtained using HUVEC (not shown). C: Endothelial cells (HUVEC) were transfected with E-selectin promoter luciferase construct together or without expression construct coding for the dominant-negative Rho GTPase RhoA (dnRhoA), RhoB (dnRhoB) and Rac1 (dnRac). After overnight incubation, cells were irradiated with a low, therapeutically relevant dose (2 Gy). After further incubation period of ~18h cells were harvested for determination of luciferase activity. Relative luciferase activity in untreated control was set to 1.0. Data shown are mean values ± sd from at least two independent experiments each performed in duplicate. Con, not transfected with dnRho forms. Fig. 6: All-trans retinoic acid (at-RA) and 9-cis retinoic acid (9c-RA) differently affect radiation-induced NF- B-regulated gene expression and E-selectin induction. A: 24h after transfection of HUVEC with NF-κB minimal promoter construct cells were exposed to different doses of γ-rays. 9-cis retinoic acid (9c-RA, 2 µM) and all-trans retinoic acid (at-RA, 2 µM) was added immediately after radiation treatment. After incubation period of ~16h (overnight), cells were harvested for determination of luciferase activity. Data shown are the mean ± sd from three independent experiments each performed in duplicate. B: 9-cis retinoic acid (9c-RA, 2 µM) and all-trans retinoic acid (at-RA, 2 µM) were added immediately after irradiation of HUVEC (2 Gy) or EA.hy-926 (10 Gy) (HUVEC and EA.hy- 926 were exposed to different doses because they differently respond to radiation (see dose response analysis shown in Fig. 1). Five hours after exposure, cells were harvested for analysis of E-selectin expression by ELISA-based method as described. Data shown are mean values ± sd from at least three independent experiments each performed in triplicate. C: 24 h after transfection of endothelial cells with E-selectin promoter construct, 9-cis retinoic acid (9c-RA, 2 µM) and all-trans retinoic acid (at-RA, 2 µM) were added. Immediately after addition of retinoic acid derivatives, cells were irradiated (HUVEC (2 Gy), EA.hy-926 (10 Gy)). After overnight incubation, cells were harvested for analysis of luciferase activity, which was set to 1.0 in the non-irradiated controls. Data shown are mean values ± sd from at least two independent experiments each performed in duplicate. 18 Downloaded from http://carcin.oxfordjournals.org/ by guest on December 18, 2012

Fig. 7: Comparative analysis of the effects of lovastatin and at-RA on radiation-induced expression of the cell adhesion molecules E-selectin and ICAM-1. Human endothelial cells (HUVEC) were pretreated or not with lovastatin (10 µM, overnight). At-RA (2 µM) was added immediately after radiation treatment. Five hours after irradiation (10 Gy), E-selectin and ICAM-1 protein expresssion were analyzed by ELISA as described in Methods. Data shown are mean values ± sd from one representative experiment performed in triplicate. Fig. 8: Lovastatin and all-trans retinoic acid impair IR-induced adhesion of human tumor cells to endothelial cells. A, B: Confluent HUVEC were pretreated or not with lovastatin (10 µM), at-RA ( 2 µM) or 9c-RA (2 µM) as described before. Afterwards, cells were irradiated (10 Gy) and adhesion of DLD1 cells was analyzed 5h later. Data shown under B are mean values ± sd from two independent experiment each performed in quadruplicate. Under A, a representative illustration of the inhibitory effect of lovastatin and at-RA on IR-stimulated tumor cell adhesion is shown. Shown is the relative adhesion to non-irradiated HUVEC. C: Effect of different doses of lovastatin (0.1-10 µM) on IR-induced (10 Gy) adhesion of DLD1 cells to HUVEC. Data shown are the mean ± sd from two independent experiments each performed in quadruplicate. Relative adhesion to non-irradiated HUVEC (=basal adhesion) was set to 1.0. D: Effect of different doses of at-RA (0.5-5µM) on IR-induced (10 Gy) adhesion of DLD1 cells to HUVEC. Data shown are the mean ± sd from two independent experiments each performed in quadruplicate. Basal adhesion to non-irradiated HUVEC was set to 1.0. Fig. 9: Effect of combined treatment with lovastatin and at-RA on radiation-induced Eselectin protein expression and tumor cell adhesion. Human endothelial cells (HUVEC) were pretreated or not with lovastatin (10 µM, overnight) before exposure to γ-rays (10 Gy). At-RA (1 µM) was added immediately after radiation treatment. Five hours after irradation, E-selectin protein expresssion (A) and tumor cell adhesion (B) were analyzed as described in Methods. Data shown are mean values ± sd from one representative experiment performed in quadruplicate. 19 Downloaded from http://carcin.oxfordjournals.org/ by guest on December 18, 2012

31420-12-1 SAWRIDGE, Indian vs. ROLAND, Twinn et al
Plenary Session 1 - 19th International C. elegans Meeting
bulletin board - Oskin et al. 2001048, Page 1 - Geological Society of ...
In tern et S ecu rity S ystem s 1 A n n u al C arrier S u m m it
Ionizing radiation-induced E-selectin gene ... - Carcinogenesis
Mechanism of Ap-1-mediated gene expression by ... - Carcinogenesis
1 Effect of inflammation on cyclooxygenase (COX ... - Carcinogenesis
1 Inhibition of Breast Cancer Growth and Invasion ... - Carcinogenesis
Fibroblasts in omentum activated by tumor cells ... - Carcinogenesis
Deletion and differential expression of p16ink4a ... - Carcinogenesis
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The candidate tumor suppressor gene NOR1 is an ... - Carcinogenesis
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