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. Author manuscript; available in PMC: 2008 Feb 28.
Published in final edited form as: J Biol Chem. 2003 Aug 12;278(43):41862–41870. doi: 10.1074/jbc.M308336200

Fig. 3. Conservation of E-boxes in the promoter of Col2a1 among human, rat and mouse and repressive effects of Snail/Slug on transcription.

Fig. 3

A. Three E-boxes were located in the promoter region of the collagen type2a1 gene in human and four E-boxes in rat and mouse. B. A comparison shows that all species have two conserved E-boxes (CAGGTG, arrow in the figure and highlightened in the table) and the relative positions in the promoter region are similar. C. Snail and Slug suppressed the transcriptional activity of the col2a1 gene promoter and/or enhancer. CAT reporter constructs are described above the graph. The percent activity relative to mock transfection was calculated. The suppressive activity was stronger with pCII-977E, which has a longer promoter region than pCII-312E. CAT constructs, pCII-977E and pCII-312 contain the collagen type2al gene fragment (−977 to +110) and (−312 to +110) respectively. Both reporter constructs had a chondrocyte-specific enhancer. Snail or Slug expression plasmid was cotransfected with CAT constructs and pSV-beta-gal as standard. CMV-CAT was a positive control. The experiment was done in triplicate. D. Snail suppressed the Col2a1 promoter activity both in differentiated and in undifferentiated ATDC5 cells in a dose-dependent manner. ATDC5 cells were cotransfected with the Snail expression plasmid, a CAT construct (pCII-977E) and pSV-beta gal. At 24 hr after transfection, the cells were put into a three-dimension culture system for 2 days to differentiate before harvesting. The activity was suppressed by the Snail expression plasmid in a dose dependent-manner (lane 1–5). ATDC5 cells were cotransfected with the Snail expression plasmid, the CAT construct without an enhancer (pCII-977) and pSV-beta gal and cultured in a monolayer for 2 days. The activity was suppressed by the Snail expression plasmid in a dose-dependent manner (lane 1–5).

The dose of Snail plasmid is 0μg (lane 1), 0.5μg (lane 2), 1.0μg (lane 3), 2.5μg (lane 4) and 12μg (or 20 lane 5). The transfection experiment was triplicated.