Fig. 6. Hth and Exd enhance repression by En in cultured cells. Drosophila S2 cells were transfected with a target gene containing a CAT reporter, binding sites that are bound co-operatively by En and Exd in vitro (oligonucleotide sequence TCGAGTCAATTAAA-TGATCAATCAATTTCG); or, as indicated in the key, the same plasmid without these sites, or with a two-nucleotide change in an Exd core binding site (oligonucleotide sequence: TCGAGTCAA-TTAAAGCATCAATCAATTTCG) and activator binding sites. Cells were harvested after 66 hours and assayed for CAT and a co-transfected reference gene. The vertical axis shows results normalized to reference gene activity, as a percentage of the maximum repression observed in each experiment. Maximum repression was eightfold for basal transcription (A) and at least tenfold for activated transcription (B,C). Error bars show the range of values from two parallel transfections. (A) The reporter (either with or without En-Exd sites, or with mutated sites) and reference plasmids were co-transfected with either a Hth expression construct, an En expression construct, or both together. Bars below the baseline indicate the slight activation seen without binding sites. (B) The reporter (with or without Exd sites mutated as indicated in the key), activator and reference plasmids were co-transfected with an En expression construct either alone or in combination with a Hth expression construct at either a low or a higher concentration as indicated (see Materials and Methods for plasmid amounts and other transfection details). There was no effect of Hth alone on activated transcription. (C) The reporter, activator and reference plasmids were co-transfected with the En expression construct (at either a low or higher concentration) either alone or with the Hth expression construct, after a prior treatment of the cells with either control or Exd dsRNA, as indicated (see key).

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