(A) HT-p21-9 cells were transduced with control lentivirus (pLKO) or lentivirus expressing shTSC2 (sequence # 10) and selected with puromycin for 5 days and then immunoblot was performed. (B) Schema: Testing the reversibility of nutlin-3a effects. (C) HT-p21-9 cells were transduced with control pLKO or shTSC2 and 5000 cells were plated in 24-well plates and, the next day, were treated with 10 uM nutlin-3a for 3 days. Then nutlin-3a was washed out and the cells were cultivated in fresh medium for 3 days and then stained for beta-Gal and microphotographed. Bars 50 um. (D) HT-p21-9 cells were transduced with control pLKO or shTSC2 (and selected for 4 days with puromycin). Then 1000 cells were plated per 60-mm dishes and, the next day, were treated with nutlin-3a for 3 days. Then nutlin-3a was washed out and cells were cultivated in fresh medium for 8 days. Colonies were stained with crystal violet.

(A)Immunoblot. WI-38tert cells were transduced with shTSC or control pLKO and cultured for 5 days. (B) WI-38tert cells were transduced with lentiviruses. Next day, medium was replaced and Nutlin (10 uM) with our without rapamycin was added. After 4 days cells were washed and stained for beta-Gal. Bars 50 um. (C) WI-38tert cells were transduced with lentiviruses. Next day, medium was replaced and Nutlin (10 uM) was added. After 4 days cells were washed and counted after 6 days.

(A) Mel-10 and Mel-9 cells were incubated with 10 uM nutlin (N) and 500 nM rapamycin (R) for 1 day and immunoblot was performed. (B) Mel-10 and Mel-9 cells were incubated with 10 uM nutlin and 500 nM rapamycin for 4 days, then drugs were washed out and cells were incubated for additional 4 days and stained for beta-Gal. Bars 50 um.

(A) Mel-10 cells were incubated with 2.5 and 10 uM nutlin with or without 500 nM rapamycin for 1 day and then immunoblot was performed. (B) Beta-Gal staining. Mel-10 cells were incubated with 10 M nutlin alone and 500 nM rapamycin for 4 days, then drugs were washed out and cells were incubated for additional 3 days and stained for beta-Gal. Bars 50 um.

) ) Immunoblot. MEF cells were incubated with 10 nutlin-3a with or without 10 nM rapamycin for 1 day and immunoblot using rabbit anti-phospho-S6 (Ser240/244) and (Ser235/236) and mouse anti-S6 was performed. (B) Beta-Gal staining. MEF cells were incubated with 10 uM nutlin alone or with 500 nM rapamycin for 4 days, then drugs were washed out and cells were incubated for additional 4 days and stained for beta-Gal. Bars 50 um.

(A) Quiescence-prone cells. Upper panel. P53 causes cell cycle arrest and inhibits the mTOR pathway, thus ensuring quiescence. Lower panel. Transduction of cells with shTSC2 activates mTOR thus converting quiescence into senescence. (B) Senescence-prone cells. Upper panel. P53 causes cell cycle arrest without inhibiting the mTOR pathway, thus ensuring senescence. Lower panel. Rapamycin inhibits mTOR thus converting senescence into quiescence.