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. 2012 Jun 26;8:589. doi: 10.1038/msb.2012.20

Figure 2.

Figure 2

PTEN status regulates glucose withdrawal-induced phospho-tyrosine induction and cell death. (A, B) Murine wild-type Pten or the lipid phosphatase inactive mutants G129E and C142S were expressed in the PTEN-null GBM cell line U87. (A) U87 cells infected with an empty vector control, wild-type Pten or the lipid phosphatase inactive mutants were starved of glucose and pyruvate for 16 and 24 h, and viability was measured by Trypan blue exclusion. Expression of wild-type Pten increased survival following glucose withdrawal by roughly two-fold (P=0.04 (*) and 0.001 (**) by Student's t-test). Error bars are standard error of the mean (n=3). (B) Western blotting with an anti-phospho-tyrosine antibody demonstrated that U87-Pten cells exhibit reduced induction of phospho-tyrosine signaling after 3 h of glucose withdrawal. Pten and GRB2 served as confirmation of Pten overexpression and equal loading, respectively. (C, D) HCT116 cells with genetic knockout of PTEN show greater cell death and phospho-tyrosine induction in response to glucose withdrawal. (C) HCT116 with wild-type PTEN (WT) or genetic knockout by homologous recombination (HCT116-22, KO) were serum starved for 16 h and then starved of glucose and pyruvate for 24 h before measurement of viability by Trypan blue exclusion. PTEN knockout cells showed reduced survival at 24 h compared with wild-type cells (P-value=0.03 (*) by Student's t-test, n=4). (D) Western blotting with an antibody against phospho-tyrosine demonstrated increased phospho-tyrosine signaling in HCT116 PTEN KO cells following 3 h of glucose withdrawal. Confirmation of loss of PTEN expression in HCT116 KO cells is shown in Supplementary Figure S4. (E, F) RWPE cells with reduced PTEN expression show greater cell death in response to glucose withdrawal (P-value=0.002 (*) by Student's t-test, n=3). (E) RWPE cells with wild-type PTEN or cells infected with an shRNA targeting PTEN were starved of glucose and pyruvate for 24 h before viability measurements by Trypan blue exclusion. Confirmation of reduced PTEN expression is shown in Supplementary Figure S4. (F) Western blotting demonstrated that RWPE cells with reduced PTEN expression demonstrate greater induction of phospho-tyrosine signaling following glucose withdrawal.