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

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

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

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 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

PAI-1 contributes strongly to radiation-induced intestinal endothelial apoptosis.

<p>High magnification of double TUNEL/CD31 staining in the villus lamina propria in Wt mice 4 hours after 19 Gy. Arrows indicate apoptotic endothelial cells. Nuclei were counterstained with DAPI (blue) (A). Representative double TUNEL/CD31 staining in Sham (A–C) and irradiated (B–D) Wt (A–B) and PAI-1 −/− (C–D) mice 5 hours after 19 Gy (B). Radiation-induced endothelial intestinal apoptosis was stronger in irradiated Wt mice than in irradiated PAI-1 −/− mice 4 hours after irradiation. Nuclei were counterstained with DAPI (blue). Quantitative assessment of TUNEL+/CD31+ cells in the villus lamina propria in Wt and PAI-1 −/− mice (C). Percentage of apoptotic endothelial cells/total endothelial cells in the villus lamina propria 4 and 5 hours after irradiation in Wt and PAI-1 −/− mice (D). Frequency of apoptotic endothelial cells in the villus lamina propria in Wt and PAI-1 −/− mice 4 and 5 hours after irradiation (E). (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

PAI-1 genetic deficiency is associated with reduced acute radiation-induced intestinal vascular injury.

<p>Intestinal vasculature was visualized by CD31 staining (red) and computerized with confocal microscopy imaging. Nuclei were counterstained with Sytox Green (A). Representative images of intestinal vasculature 24 hours after irradiation in Wt and PAI-1 −/− mice (B). Quantitative assessment of vasculature integrity 24 hours after 19 Gy (C). (n = 6 mice/group) # p<0.05.</p