Fig. 5. Nucleostemin depletion triggers G2-M arrest and apoptosis. Doxycycline (Dox)-inducible nucleostemin-knockdown (shNS) U2OS and H1299 cells and their respective controls (shScr) were created (see supplementary material Fig. S4). (A) U2OS-shNS cells displayed a 47%, 57% and 67% loss of nucleostemin protein after 4, 7 and 10 days of Dox (20 µg/ml) treatment, respectively. Nucleostemin depletion reduces MDM2 levels without changing the amount of p53. p53 transcriptional activity, as determined by the protein levels of two of its transcriptional targets, p21 and Bax, and cleaved caspase-3 (aCas3) were increased. Tub, -tubulin. (B) Population-doubling levels (PDL) and time (1/PDL in days) were measured over a 6 day period. The doubling time of Dox-treated U2OS-shNS cells was significantly prolonged compared with that in shScr cells and untreated shNS cells (black bars). The PDL of H1299 cells was unchanged by nucleostemin knockdown (grey bars). (C) Cell-cycle analyses showed decreased G1-G0 and increased G2-M and sub-G1 cell percentages in the nucleostemin-knockdown U2OS cells. (D) The percentage of prophase cells labeled with anti-phospho-Histone3 (pH3) is reduced by nucleostemin knockdown. (E) Binding between the endogenous nucleostemin and MDM2 increases in M-phase-synchronized cells.