TSC-22 induces p53 expression.

<p>(<b>A</b>) TSC-22 and p53 cDNA constructs were cotransformed into EGY48 yeast cells to test for protein–protein interaction within the yeast two-hybrid system. Transformants were assayed for their ability to grow on medium lacking leucine (left) and for β-galactoside expression (right). (<b>B</b>) <i>HeLa</i> cells and <i>Caski</i> cells were infected with Ad-<i>TSC-22</i> for the indicated times. Protein levels were analyzed by Western blot with the DO-1 antibody against p53, the anti-TSC-22 antibody, and the anti-β-actin antibody as a loading control. (<b>C</b>) <i>HeLa</i> cells were transfected with 1 µg of Flag-TSC-22 expression vector. 24 h post-transfection, p53, Puma, p21, and TSC22 expression were analyzed by Western blotting and semi-quantitative RT-PCR with protein and total RNA obtained from each cell line. (<b>D</b>) <i>HeLa</i> cells stably expressing shRNA specific for TSC-22 and non-targeting control shRNA were analyzed to determine the protein and mRNA expression levels of p53 and Puma. (<b>E</b>) Luciferase activity of the p53RE (responsible element)-driven promoter were assessed with transfection of the indicated amount of Flag-TSC-22 plasmid in <i>HeLa</i> cells (upper panel). Activity of the p53RE-promoter was assessed in <i>sh-con</i> and <i>sh-TSC-22</i> expressing <i>HeLa</i> cells (lower panel) by luciferase assays. (<b>F</b>) One µg of Flag-TSC-22 or Flag-mock vector was transfected into <i>p53</i>^+/+ or <i>p53</i>^−/−<i>HCT116</i> cells. At 48 h post-transfection, cell lysates were analyzed by Western blotting with the indicated antibodies. (<b>G</b>) Stability of p53 protein was assessed in <i>HeLa</i> cells infected with Ad-<i>TSC-22</i> or Ad-<i>LacZ</i>. 24 h after infection, cells were treated with cycloheximide (50 µg/mL) for the indicated periods of time. Cell lysates were analyzed by Western blotting with anti-p53 antibody (DO-1) with β-actin as a loading control.