Research Paper Volume 2, Issue 6 pp 344—352

Figure 1. Depletion of TSC2 converts quiescence into senescence in HT-p21-9 cells. (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.