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. 2014 Dec 15;6(2):617–632. doi: 10.18632/oncotarget.2710

Figure 7. Decrease of endogenous G2E3 levels after DNA damage.

Figure 7

(A) G2E3 mRNA levels are decreased upon cisplatin treatment. U2OS, HCT116 p53+/+ and HCT116 p53−/− cells were transfected with control-siRNA and either left untreated or treated with 30 μM cisplatin for 16 h. Cells were harvested and G2E3 mRNA levels were analyzed by quantitative RT-PCR and normalized to the reference gene GAPDH. Data are represented as mean. Error bars represent the standard deviation (SD, n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 (Student's t-test). (B) G2E3 mRNA levels are reduced by gemcitabine treatment. U2OS cells were treated with 300 nM gemcitabine for 6, 16 or 24 h. The cells were harvested after a total incubation time of 48 h. G2E3 mRNA levels were analyzed as in A. (C) Endogenous G2E3 protein levels are decreased after DNA damage. U2OS cells were either left untreated or treated with 30 μM cisplatin for 16 h, and during the last 4 h with 20 μM of the proteasome inhibitor MG132. G2E3 was immunoprecipitated, followed by immunoblot detection of G2E3. β-Galactosidase (β-Gal) was used as control antibody. As a further control, lysis buffer without cell lysate was incubated with the indicated antibodies. (D) HA-G2E3 protein levels are reduced by cisplatin treatment. U2OS cells were transiently transfected with a plasmid encoding HA-G2E3 or with an empty plasmid (pcDNA3) as control, followed by treatment with 30 μM cisplatin for 16 h as indicated. Cell lysates were analyzed by immunoblotting and detection using antibodies to the indicated proteins. Cotransfection of a GFP expression plasmid served as control for transfection efficiency. (E) Model of G2E3 affecting cell survival. G2E3 is down-regulated in a DNA damage-responsive manner. On the other hand, G2E3 supports cell survival. In part, this pro-survival function is carried out by sustaining the ATR-Chk1 signaling pathway, thereby avoiding replicative stress.