Effect of irradiation on mRNA and protein expression of SMAD3, PAI-1 and SMAD co-repressors and inhibitors in HUVEC.

<p>(A) Irradiation induces SMAD pathway and PAI-1 in HUVEC cells. mRNA levels of SMAD3 and PAI-1 in HUVEC 6, 24, 48 and 72 hours after irradiation were measured by real time PCR. mRNA level of GAPDH was used as housekeeping gene and value 1 was assigned to unirradiated cells for each time. Protein levels of Smad3, phospho-Smad3 and PAI-1 were followed by western blot. (B) Radiation dose- and time-dependent mRNA level and protein abundance of TGIF1, SnoN, Ski and Smad7 were measured by real time PCR and western blot. Results are the mean ± SEM of 3 independent experiments realized in triplicates. *P<0.05 vs unirradiated cells.</p

Kinetic analyses of mRNA levels of SMAD co-repressors and inhibitors in a mouse model of 19 Gy-localized small intestinal irradiation: mRNA levels of TGIF1, SnoN, Ski and SMAD7 were measured by real time PCR in total intestinal tissues, 5 hours, 1 day, 3 days, 14 days and 42 days after irradiation.

<p>mRNA level of 18S was used as housekeeping gene and dot line indicates value 1 assigned for control sham-operated animals at each time. Results are +/- SEM (n = 6 to 8 mice/group). *p<0.05 versus sham mice.</p

TGIF1 has no effect either on radiation-induced Smad pathway activation or on radiation-induced PAI-1 overexpression in endothelial cells.

<p>(A) HUVECs were transfected with (CAGA)9-Luc reporter plasmid for 24 h and were serum starved for 18 hours. Cells were irradiated at 10 Gy in presence or not of 10 ng/mL TGF-β1. Relative luciferase activity was measured 24 h after irradiation (B) HUVECs were co-transfected for 24 h with CAGA9-Lux and myc-Smad3 with or without myc-TGIF1. Relative luciferase activity was measured 24 h after irradiation and/or treatment with 10 ng/mL of TGF-β1. (C) HUVECs were transfected with wt-PAI-1 Luc reporter or CAGA box-mutated PAI-1 Luc reporter (Δ123-PAI-1 Luc) and myc-Smad3 with or without myc-TGIF1. Luciferase activity was assayed 24 hours after irradiation. Relative luciferase activity is the mean ± SEM of at least 3 independent experiments realized in triplicates (D) HUVECs were transfected for 24 h with myc-Smad3 with or without myc-TGIF1. Cells were irradiated at 10 Gy and PAI-1 expression was measured 24 h after irradiation. E-G) HUVECs were transfected with non–targeting siRNA (siRNA control) or siRNA TGIF1 for 24 h and irradiated at 10 Gy. Silencing efficiency was confirmed by western-blot (E). mRNA level (F) and protein levels of PAI-1 and Phospho-Smad3 were performed by western-blots (G). Results are the mean +/- SEM of two independent experiments realized in triplicates and representative blots from three independent experiments are shown. *p<0.05</p

TGIF1 genetic deficiency sensitizes mice to gastrointestinal syndrome and radiation enteropathy.

<p>Kaplan-Meier analyses represent the percent survival of TGIF1^+/+, TGIF1^+/− and TGIF1^−/− mice following (A) 13 Gy total body irradiation and (B) 19 Gy localized small intestinal irradiation. Statistical differences were determined by the log rank test, *p = 0.0033, **p = 0.19, ***p = 0.00065, ^#p = 0.06, ^##p = 0.11, ^###p = 0.000014.</p

PAI-1 influences the pro-survival Akt pathway in murine endothelial cells.

<p>Representative western blots of phospho and total Akt in Wt and PAI-1 −/− ECs (A) and in Wt and PAI-1 −/− ECs platted on different coatings 24 hours after 20 Gy (B). Representative western blots of Phospho p38 MAPK, Phospho ERK1/2, Phospho PDK-1, Phospho PTEN and total PTEN in Wt and PAI-1 −/− ECs 24 hours after 20 Gy (C). Percentage of active PTEN in Wt and PAI-1 −/− ECs 24 hours after 20 Gy (D). All experiments were realized in triplicates.</p

PAI-1 influences the pro-survival Akt pathway in human endothelial cells by PTEN inactivation.

<p>Representative western blots of phospho-Akt, total Akt, phospho-PTEN, total PTEN, phospho-p38MAPK, p65/RelA, phospho-ERK1/2, phospho-PDK-1, Bcl-2 and Bcl-XL in HUVECs transfected or not with siRNA PAI-1 24 hours after 20 Gy (A). Representative western blots of phospho and total Akt in HUVECs that stably overexpress PAI-1. Akt activation is reduced in human ECs that overexpress PAI-1 (B). Silencing efficiency determined by western blot in HUVECs transfected with siRNA PTEN or siRNA PDK-1 (C). Representative images of HUVECs knocked-down for PTEN, PDK-1, PAI-1, PTEN/PAI-1, or PDK-1/PAI-1 24 hours after 20 Gy (D). Representative western blots of phospho-Akt, total Akt, p65/RelA, phospho-ERK1/2, PTEN, BCL-2 and BCL-XL in HUVECs transfected with siRNA PTEN, PDK-1 and/or siRNA PAI-1 24 hours after 20 Gy(E) .</p

TGIF1 genetic deficiency is not associated with modifications of radiation-induced TGF-β pathway-related molecular profile <i>in vivo</i>.

<p>mRNA levels of (A) TGF-β signaling-related genes and (B) TGF-β/Smad target genes in TGIF1^+/+, TGIF1^+/− and TGIF1^−/− mice 3 days after 19 Gy localized small intestinal irradiation were determined by real time PCR. mRNA level of 18S was used as housekeeping gene and value 1 was assigned to sham-operated TGIF1^+/+ animals. Results are the mean ± SEM with 8 to 10 mice per group. *P<0.05 vs TGIF1^+/+ sham mice. ^#P<0.05 vs TGIF1^+/+ irradiated mice. No significant difference was obtained between TGIF1^+/+, TGIF1^+/− and TGIF1^−/− sham-irradiated animals.</p

Radiation induces activation of the TGF-β pathway in a mouse model of 19 Gy-localized small intestinal irradiation: mRNA levels measured by real time PCR of TGF-β1, SMAD3 and PAI-1 in total intestinal tissues, 5 hours, 1 day, 3 days, 14 days and 42 days after irradiation.

<p>mRNA level of 18S was used as housekeeping gene and dot line indicates value 1 assigned for control sham-operated animals at each time. Results are +/− SEM (n = 6 to 8 mice/group). *p<0.05 versus sham mice.</p

PAI-1 overexpression is associated with increased radiation sensitivity of endothelial cells.

<p>Representative western blot (A) and quantification of PAI-1 protein expression of five clones of HUVECs that stably overexpressed PAI-1 (B). Clonogenic assay in control HUVECs and clones 1, 3 and 5 (C). Surviving fraction after 2 Gy (D). Results are the mean +/− SEM (n = 6 per conditions) * p<0.05 versus control HUVECs.</p

PAI-1 contributes slightly to radiation-induced intestinal epithelial cell apoptosis in crypts.

<p>High magnification of double TUNEL/E-cadherin staining in crypts in Wt mice 5 hours after 19 Gy. Arrows indicate apoptotic epithelial cells. Nuclei were counterstained with DAPI (blue) (A). Representative double TUNEL/E-cadherin staining in Sham (A–C) and irradiated (B–D) Wt (A–B) and PAI-1 −/− (C–D) mice 5 hours after 19 Gy. (B). Quantitative assessment of TUNEL+/E-cadherin+ cells in crypts in Wt and PAI-1 −/− mice 4, 5 and 24 hours after irradiation (C). Radiation-induced epithelial cell apoptosis in crypts was stimulated in both types of mice. The number of apoptotic epithelial cells was higher in Wt mice than in PAI-1 −/− mice only 5 hours after irradiation. (n = 6 mice/group) # p<0.05 versus sham mice with the same genotype. * p<0.05 between irradiated Wt and PAI-1 −/− mice.</p