Modulations of the cell cycle change the transcription of Snail1a, the direct downstream target of p65. (A) p65 activates the CAT reporter gene driven by the Snail1a promoter. The following combinations were injected, and CAT activities were assayed at the shield stage: (1) Sn1 (Snail1a promoter region including the NF-κB–binding site)-CAT+ConMO, (2) Sn1-CAT+p65MO, (3) Sn1-CAT+GemMO, (4) Sn1-CAT+p65 mRNA, (5) Sn1Mut (Snail1a promoter region with the NF-κB–binding site mutated)-CAT+ConMO, (6) Sn1Mut-CAT+p65MO, (7) Sn1Mut-CAT+GemMO, and (8) Sn1Mut-CAT+p65 mRNA. The y axis indicates the absorbance of the sample at 405 nm. Error bars indicate the standard error of six individual tests. (B) The Snail1a promoter region containing the NF-κB–binding site, but not a 1-kb downstream control region, was identified to associate with S276-p65 by the ChIP assay. PCR using sheared chromatin before immunoprecipitation and immunoprecipitation using preimmune serum served as positive and negative controls, respectively. (C–M) Modulations of the cell cycle change the transcription of Snail1a through NF-κB. Note that Snail1a was ectopically expressed in embryos injected with geminin mRNA (E), Cdt1MO (G), or MCM5MO (I), whereas it was reduced in embryos injected with GemMO (D), Cdt1 mRNA (H), or p21MO (J). Injection of Gem* mRNA was ineffective on the Snail1a transcription at the blastoderm margin (F). The ectopic expression of Snail1a caused by geminin mRNA was rescued by the coinjection of p53MO (K). Both p65MO alone (L) and geminin mRNA plus p65MO (M) led to a dramatic reduction of Snail1a. The shield stage embryos were viewed from the lateral with the shield to the right. Bar, 100 µm.