(A) Analysis of mRNA expression by semi–qRT-PCR in human mammary epithelial cells (HMEC) and human umbilical vein epithelial cells (HUVEC) expressing constitutively active Notch1 (Notch1ICD) or Notch4 (Notch4ICD)

(B) Analysis of mRNA expression by qRT-PCR in HMEC expressing Notch1ICD. Results are normalized to the vector control ( = 3). *, P < 0.05. Error bars show SEM. (C) Immunoblots for Slug and Snail in HMEC, HUVEC, and human aortic endothelial cells (HAEC) transduced with Notch1ICD, Notch4ICD, or the empty vector. (D) HMEC expressing Jagged1 or Dll4 were cocultured with parental HMEC and immunoblotting for Slug was performed.<p><b>Copyright information:</b></p><p>Taken from "Slug is a direct Notch target required for initiation of cardiac cushion cellularization"</p><p></p><p>The Journal of Cell Biology 2008;182(2):315-325.</p><p>Published online 28 Jul 2008</p><p>PMCID:PMC2483533.</p><p></p

(A) Phase contrast (left) and DAPI (right) images of AV canal explants from wild-type and embryos

Bars, 250 μm. (B) Quantitative analysis of EMT in AV canal explants from E9.5 wild-type (wt), , and embryos after 48 h in culture. Results represent the distance of a positive pixel (DAPI-stained nucleus) to the closest point of the AV canal normalized to the area of the AV canal tissue. *, P < 0.05. (C) expression in the AV canal explant assay as visualized by β-galactosidase staining of wild-type and AV explants. The rounded morphology of most of the LacZ cells is shown on the right. The black square indicates the region of higher magnification shown to the right. Bars, 50 μm. (D) Representative sections of wild-type and hearts counterstained with Nuclear Fast Red used for analysis in E. Dotted blue lines highlight the superior and inferior AV cushions. Bars, 50 μm. (E) Quantitation of the cellularity of the superior and inferior cushions in E9.5 wild-type (wt; = 3) and ( = 3) embryos. Error bars show SEM. (F) BrdU analysis on the percentage of proliferating cells in wild-type ( = 4) and ( = 6) AV canal cardiac cushions (total), the AV canal endocardium (Endo), and AV canal mesenchymal cells (Mesen; 10–15 sections per embryo). Error bars show SEM. (G) Vector- or Slug-transduced HMEC were subjected to an endothelial wounding assay. Bars represent the distance migrated after 24 h ( = 4). *, P < 0.05. Error bars show SD. (H) Vector- or Slug-transduced HMEC were evaluated in a modified Boyden chamber assay with 20 ng/ml PDGF-BB present in the lower chamber. Bars represent the total number of cells migrated after 4 h ( = 6). *, P < 0.05. Error bars show SD.<p><b>Copyright information:</b></p><p>Taken from "Slug is a direct Notch target required for initiation of cardiac cushion cellularization"</p><p></p><p>The Journal of Cell Biology 2008;182(2):315-325.</p><p>Published online 28 Jul 2008</p><p>PMCID:PMC2483533.</p><p></p

(A) qRT-PCR analysis demonstrating efficient knockdown of in HMEC with two different shRNAs targeting (shCSL) compared with a random control sequence (shRan)

(B) qRT-PCR of and in vector- or Dll4-activated HMEC transduced with shCSL constructs ( = 3). *, P < 0.05 vector shRandom versus HA-D114 shRandom; **, P < 0.05 HA-D114 shRandom versus HA-D114 shCSL-A or shCSL-B. (C) Immunoblotting for Slug, VE-cadherin, and CD31 in vector- or Dll4-activated HMEC transduced with shCSL or shSlug constructs. (D) qRT-PCR of vector- or CSL-VP16–expressing HMEC for and ( = 3). *, P < 0.05. (E) PCR after ChIP with anti–FLAG-M2 antibody on HMEC-expressing vector (vec) or FLAG-CSL (CSL) to demonstrate CSL binding to the human promoter. The negative (-ve) control represents PCR of the promoter after ChIP using FLAG-M2. (F) EMSA using nuclear lysates collected from vector- or FLAG-CSL–expressing HMEC and P-labeled double-stranded oligonucleotides spanning each of the two CSL binding sites in the human promoter. Supershift assays with anti–FLAG-M2 or IgG control antibodies, and competition assays with 50× wild-type (wt) or mutant probes are also shown. Error bars show SEM.<p><b>Copyright information:</b></p><p>Taken from "Slug is a direct Notch target required for initiation of cardiac cushion cellularization"</p><p></p><p>The Journal of Cell Biology 2008;182(2):315-325.</p><p>Published online 28 Jul 2008</p><p>PMCID:PMC2483533.</p><p></p

(A) β-galactosidase staining (representing expression) of whole-mounted hearts from embryos from E9

5 to 11.5. Arrows point to the AV canal or OFT at E11.5. Bars, 250 μm. (B) Sections through the AV canal and OFT of β-galactosidase–stained hearts from E9.5 to 11.5. Bars, 100 μm. (C) Immunofluorescence staining for Slug (red) and CD31 (green) in E11.5 embryonic mouse hearts. Arrowheads point to cells coexpressing Slug and CD31. Bars, 25 μm. (D) In situ hybridization for and in a 65-d human embryonic heart. Arrows point to the mitral and tricuspid valves, arrowheads indicate the interatrial septum, and the asterisk marks the AV septum. A higher magnification image of the heart valve is shown in the right panel of each analysis. Bars, 1 mm.<p><b>Copyright information:</b></p><p>Taken from "Slug is a direct Notch target required for initiation of cardiac cushion cellularization"</p><p></p><p>The Journal of Cell Biology 2008;182(2):315-325.</p><p>Published online 28 Jul 2008</p><p>PMCID:PMC2483533.</p><p></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

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 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