Fig. 5.
Fig. 5. Functional cooperation between KLF6 and Sp1 in transactivating endoglin and other GC box promoters. / (A) Diagram with the pCD105(−50/+350) reporter construct that contains the proximal region of the endoglin promoter. The sequence corresponding to the −50/+1 fragment includes the putative binding motifs for Smad (SBE), Sp1, and KLF6, as indicated. (B) Effect of mutation at −37/−29 of the endoglin promoter. Schneider-2 Drosophila cells were transfected with either the wild-type (WT) pCD105(−50/+350) reporter construct or the corresponding version containing a mutation in the GC box motif (MUT), in the presence or absence of the KLF6 expression vector(pPAC-KLF6), as indicated. Transcriptional activity was measured 24 hours later by the luciferase assay and was plotted as relative luciferase units (RLU). One of 4 representative experiments that yielded similar results is shown, with SD indicated. (C)Drosophila SL-2 cells were transiently transfected with 1 μg endoglin promoter-reporter construct pCD105(−50/+350), combined with the indicated amounts of expression plasmids for KLF6 and Sp1. Luciferase activity was measured after 24 hours. KLF6/Sp1-transfected–sample fold induction values are referred to the corresponding sample transfected only with an empty vector, whose arbitrary value is 1. Shown is 1 of 4 representative experiments whose results were similar, with the means (± SD shown). (D-H)Drosophila SL-2 cell cultures grown on 35-mm dishes were cotransfected with a combination of the indicated amounts ofSp1-pAC and KLF6-pAC expression vectors plus 500 ng (D). GC3-Luc (artificial promoter containing GC boxes). (E) phTG5luc (TGF-β1 promoter). (F)pUK-Luc (uPA promoter). (G) pTβRIP-Luc (−867 to −228; TβRI promoter). (H) pGL-Col 3 (collagen α1(I) promoter), as described in “Materials and methods.” After a 48-hour incubation, cell lysates were prepared, and luciferase activity in each lysate was determined and expressed as -fold increase. Each value represents the average ± SD from triplicate determinations. Each experiment was repeated 3 times with similar results, and representative results are shown. In all promoter contexts, a cooperative transactivation is seen between KLF6 and Sp1.

Functional cooperation between KLF6 and Sp1 in transactivating endoglin and other GC box promoters.

(A) Diagram with the pCD105(−50/+350) reporter construct that contains the proximal region of the endoglin promoter. The sequence corresponding to the −50/+1 fragment includes the putative binding motifs for Smad (SBE), Sp1, and KLF6, as indicated. (B) Effect of mutation at −37/−29 of the endoglin promoter. Schneider-2 Drosophila cells were transfected with either the wild-type (WT) pCD105(−50/+350) reporter construct or the corresponding version containing a mutation in the GC box motif (MUT), in the presence or absence of the KLF6 expression vector(pPAC-KLF6), as indicated. Transcriptional activity was measured 24 hours later by the luciferase assay and was plotted as relative luciferase units (RLU). One of 4 representative experiments that yielded similar results is shown, with SD indicated. (C)Drosophila SL-2 cells were transiently transfected with 1 μg endoglin promoter-reporter construct pCD105(−50/+350), combined with the indicated amounts of expression plasmids for KLF6 and Sp1. Luciferase activity was measured after 24 hours. KLF6/Sp1-transfected–sample fold induction values are referred to the corresponding sample transfected only with an empty vector, whose arbitrary value is 1. Shown is 1 of 4 representative experiments whose results were similar, with the means (± SD shown). (D-H)Drosophila SL-2 cell cultures grown on 35-mm dishes were cotransfected with a combination of the indicated amounts ofSp1-pAC and KLF6-pAC expression vectors plus 500 ng (D). GC3-Luc (artificial promoter containing GC boxes). (E) phTG5luc (TGF-β1 promoter). (F)pUK-Luc (uPA promoter). (G) pTβRIP-Luc (−867 to −228; TβRI promoter). (H) pGL-Col 3 (collagen α1(I) promoter), as described in “Materials and methods.” After a 48-hour incubation, cell lysates were prepared, and luciferase activity in each lysate was determined and expressed as -fold increase. Each value represents the average ± SD from triplicate determinations. Each experiment was repeated 3 times with similar results, and representative results are shown. In all promoter contexts, a cooperative transactivation is seen between KLF6 and Sp1.

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