Notch signaling regulates Slug expression through a CSL-dependent pathway. (A) qRT-PCR analysis demonstrating efficient knockdown of CSL in HMEC with two different shRNAs targeting CSL (shCSL) compared with a random control sequence (shRan). (B) qRT-PCR of Slug and HeyL in vector- or Dll4-activated HMEC transduced with shCSL constructs (n = 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 Slug and HeyL (n = 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 Slug promoter. The negative (-ve) control represents PCR of the ZNF3 promoter after ChIP using FLAG-M2. (F) EMSA using nuclear lysates collected from vector- or FLAG-CSL–expressing HMEC and ³²P-labeled double-stranded oligonucleotides spanning each of the two CSL binding sites in the human Slug 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.