Figure 2. Gerosuppression and mTOR inhibition.

A. Geroconversion. Torin 1 and PP242 were added to IPTG-treated HT-p21 cells, as shown in Figure 1A. Cells plated at low density were treated with IPTG in the presence of serial dilutions of inhibitors. After 3 days, IPTG and inhibitors were washed out and cells were incubated in drug-free medium. Then colonies were stained with Crystal Violet and counted. Data are mean number of colonies per a sector of a well ± SD. B. Immunoblot analysis. HT-p21 cells were treated with IPTG in combination with Torin 1 or PP242 for 24 h and lysed. Rapamycin (R) at 500 nM was included as additional control. Immunoblotting was performed with the indicated antibodies. Note: IPTG-induced p21 is highly expressed in all samples, confirming that the inhibitors do not interfere with p21 induction by IPTG and cell cycle arrest.