FIG. 7.

mTOR regulates p73 levels and activity. (A) mTOR knockdown induces p73. MDA-MB-231 cells were transduced with lentivirus engineered to express shRNA against the FRAP1 subunit of mTOR or with control lentivirus. Protein lysates were harvested 3 days after transduction, and mTOR, p73, p4EBP1, and actin were analyzed by Western blotting to demonstrate knockdown of mTOR levels and activity and induction of p73 levels. Western blots are representative of at least three independent experiments. (B) MDA-MB-231 cells were transduced with lentivirus engineered to express shRNA against GFP or p73. Protein lysates were harvested 5 days after transduction, and reduction of p73 levels was confirmed by Western blotting. (C) p73 activity is induced in MDA-MB-231 cells treated with 20 nM rapamycin, with or without concurrent serum starvation, and the result was verified using p73 RNAi. Cells were serum starved by preincubation in serum-free medium overnight before treatment with rapamycin. p73 RNAi was performed by transducing cells with lentivirus engineered to express shRNA against either GFP or p73 72 h before treatment. Total RNA was purified 48 h after treatment and reverse transcribed, and quantitative real-time PCR was performed with primers for the indicated genes. The samples were normalized to GAPDH, and the results are presented as increase over vehicle control values for an average of three experiments. Samples that exhibited a 30% or greater increase relative to control are indicated in red. Twelve of 17 genes exhibited a p73-dependent increase in RNA levels after rapamycin treatment and serum starvation. (D) Change in RNA as in panel C for INSR, TSC1, and XDH shows rapamycin/serum starvation-induced changes that are p73 dependent. Error bars represent standard deviations for three experiments. (E) Semiquantitative ChIP was performed to assess levels of p73 binding to genomic regions in INSR, TSC1, and XDH promoters or introns in Rh30 cells treated with vehicle or 40 nM rapamycin for 24 h. (F) MDA-MB-231 cells were transduced with shRNA lentivirus as for panel B and treated with 20 nM rapamycin or vehicle. At the indicated times cells from treated or control cultures were counted, and changes in cell growth rates due to p73 and rapamycin are shown. Error bars represent standard deviations from three experiments.