Figure 2

Determination of a core responsive element of SOX11 in the GDF5 promoter. (A) Levels of Gdf5 mRNA in ATDC5 and C3H10T1/2 cells retrovirally transfected with the control vector (Rx-EV) or the vector expressing human SOX11 (Rx-SOX11). mRNA levels were determined by quantitative RT-PCR, normalized to Gapdh, and expressed as means (bars) ± SDs (error bars) for 4 wells/construct. *P < 0.05 vs. Rx-EV. (B) Gdf5 and Sox11 mRNA levels in ATDC5 cells retrovirally transfected with the shRNA vector targeting GFP (Rx-shGFP) or targeting mouse Sox11 (Rx-shSox11). *P < 0.05 vs. Rx-shGFP. (C) Deletion analysis using constructs lacking putative SOX-family-binding motifs in the proximal human GDF5 promoter (+151/+276) in ATDC5, C3H10T1/2, and HeLa cells. Five putative SOX family binding sites are shown (+159 to +165, +179 to +185, +222 to +228, +238 to +244, +258 to +264). The site with 1 base difference from the ideal binding site is shaded in dark gray, and those with 2 bases difference are indicated in light gray. Luciferase assays were carried out as above, in ATDC5, C3H10T1/2 and HeLa cells. *P < 0.05 vs. deletion of +151/+214 **P < 0.05 vs. deletion of +151/+229. (D) Identification of a core responsive element of SOX11 in the GDF5 promoter (segment A: +209/+257). Site-directed mutagenesis analysis with 6 mutated constructs (M1–M6). Base changes for each mutation as shown. Luciferase assays were carried out as above, in ATDC5, C3H10T1/2 and HeLa cells. The data for the luciferase assay are expressed as means (bars) ± SDs (error bars) of Relative luciferase activity for 4 assays/construct. *P < 0.05 vs. wild type promoter, –448/+319.