(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) 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) 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