KCTD11 promoter is strongly activated by Sp1 TF. (A) D. Mel-2 cells (Invitrogen) were cultured in Drosophila Serum-Free Medium (Gibco Life Technologies) plus18 mM L-glutamine (Sigma) at room temperature. KCTD11-Luc reporter constructs (1 μg) and pPac-LacZ (200 ng) were co-transfected along with 1 μg of pPac empty vector, pPac-Sp1 or pPac-Sp2 (left panel) or pPac-Sp3 and pPac-Sp1+pPac-Sp3 (right panel) using Cellfectin (Invitrogen). After 48 h, the cells were harvested and the luciferase activity was assayed using the Single Luciferase Assay System (Promega), normalized to β-galactosidase activity using β-Galactosidase Enzyme Assay System (Promega). Each experiment has been done in triplicates. Values are the means ± S.D. (B) EMSAs showing the binding of Sp1 to KCTD11 promoter. Whole extracts were prepared from 293T HEK cells transfected with pCDNA-Sp1 (12 μg), using buffer C . Cell extracts were incubated in vitro with 32P-labeled KCTD11-Sp1 probes (Sp1-A-F) or 32P-labeled canonical Sp1 probe [additional file 2]. Binding specificity was evaluated by competition with an excess (100x) of the cold probe or with a non-specific probe. For the supershift assays, the proteins were pre-incubated with two different anti-Sp1 antibodies (Santa-Cruz) at 4°C for 30 min. (C) ARO (thyroid cancer) and HCT15 (colon cancer) cell line were cultured, respectively, in RPMI-1640 and DMEM supplemented with 10% FCS. Chromatin Immunoprecipitations (ChIP) were performed by using the following antibodies: anti-Sp1 (1C6) X (sc-420; Santa Cruz Biotech), anti-Sp3 (F-7) X (sc-28305; Santa Cruz Biotech), anti-Acetyl-Histone3 (Cell Signaling). Eluted DNA has been analyzed with real-time q-PCR, normalized to GAPDH (left panels). Bars represent the mean of 3 independent experiments ± SD (*, p < 0.05, HCT15 versus ARO). Total protein levels of Sp1 and Sp3 were analyzed by Western blot (right panel).