Fig. 3

The T-element at -121 bp of the p21 promoter mediates repression by TBX3 and TBX3 binds this region in vitro and in vivo. a Alignment showing conservation of the T-element residue at -121 bp (T-121 bp) of the p21 promoter across a number of different species. b ATDC5 and SW1353 cells were co-transfected with pGL3 basic empty, WTp21 or Mutp21 luciferase reporter constructs together with pCMV empty or WT TBX3. The plasmid pRL-TK containing the Renilla luciferase reporter gene was also introduced to normalize transfection efficiency. Fold repression was obtained by comparing the results to that of the empty pCMV vector transfection. The values indicate the mean of three independent experiments ± SEM (*p < 0.05; **p < 0.01, ***p < 0.001). c DNA-affinity immunoblot assay. Lysates from SW1353 cells were incubated with a biotinylated probe matching the sequence of the wild-type or mutated T-element at position -121 bp of the p21 promoter. A pull-down was performed using streptavidin magnetic beads and the lysates run on an 8 % SDS-PAGE gel and analysed by western blotting using an antibody to TBX3. d SW1353 control and shTBX3 lysates were used in a ChIP assay performed with antibodies against TBX3 or IgG (negative control). Immunoprecipitated DNA was assayed by qRT-PCR with primers against the p21 promoter or GAPDH (negative control). The values indicate the mean of two independent experiments each performed in triplicate ± SEM (***p < 0.001)