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Fig. 5 | Cell & Bioscience

Fig. 5

From: Multiple Fra-1-bound enhancers showing different molecular and functional features can cooperate to repress gene transcription

Fig. 5

Requirement upon p300/CBP activity for eRNA expression at Fra-1-bound TGFB2 enhancers. A Dependence of p300/CBP presence for TGFB2 mRNA and eRNAs expression in MDA-MB-231 cells. Cells were RNAi-depleted in both p300 and CBP. siCTL-transfected cells served as a reference. 72 h later, protein, mRNA and eRNA levels were analyzed. Left panel: Immunoblotting of p300 and CBP down-regulation. Fra-1 level was also assessed and GAPDH was used as an invariant control. Middle and right panels: TGFB2 mRNA and eRNA levels, respectively. S26 mRNA was used as an invariant internal standard. The data are the mean of 4 independent experiments in which values were normalized to that of the siCTL condition set to 1 for each amplicon. B Dependence upon the activity of p300/CBP for expression of eRNAs at Fra-1-bound TGFB2 enhancers. MDA-MB-231 cells were transfected with siCTL to allow comparison with the experiments shown in Fig. 5D. 56 h later, A485 or DMSO were added for 16 h and TGFB2 mRNA or eRNAs were RT-qPCR-assayed. Left panel: immunoblotting of H3, H3K27ac, p300, CBP and Fra-1 with GAPDH used as an invariant control. Middle and right panels: TGFB2 mRNA and eRNA levels, respectively. S26 mRNA was used as an invariant standard. The data are the mean of 7 independent experiments in which values were normalized to the DMSO condition set to 1 for each amplicon. C Dependence upon Fra-1 for recruitment of p300/CBP at the TGFB2 enhancers. Cells were transfected with siCTL or siFra-1 for 72 h before p300/CBP ChIP-qPCR assays. The data are the mean of 5 independent experiments in which values were normalized to that of amplicon + 32 under control condition arbitrarily set to 1. D Dependence upon the KAT activity of p300/CBP for TGFB2 mRNA and eRNA expression levels after RNAi-mediated depletion of Fra-1. MDA-MB-231 cells were transfected with either siCTL or siFra-1 for 72 h, with A485 or DMSO added for the last 16 h. Left panel: immunoblotting of Fra-1, p300, CBP, histone H3 and H3K27ac, with GAPDH as an invariant control. Middle and right panels: RT-qPCR assays of TGFB2 mRNA and eRNA levels, respectively, using S26 mRNA as an invariant standard. The data are the mean of 7 independent experiments in which the values were normalized to that of amplicon + 32 under control condition arbitrarily set to 1

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Cell & Bioscience

ISSN: 2045-3701

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