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. Author manuscript; available in PMC: 2015 Jan 1.
Published in final edited form as: J Neurooncol. 2013 Oct 27;116(1):89–97. doi: 10.1007/s11060-013-1283-3

Fig. 2. LY294002 and 2ME2 demonstrate synergy in blocking HIF1α accumulation in GBM cell lines.

Fig. 2

(A) Immunoblot showing pAKT and pERk levels in U373MG-Null and U373MG-PTEN cell lines treated with 25μM of 2ME2 either alone or in combination with 10 μM of LY294002. Cells were incubated with indicated conc. of drug for 16 hrs followed by lysate preparation and Western blot analysis using pAkt, pErk, Akt and Erk antibodies. (B) Immunoblot showing synergy between 2ME2 and LY294002 blockade in LN229 vIII cells treated with indicated amount of drug for 16 hrs.(C) U87MG -Null and U87MG-PTEN cell lines were treated with 25 μM 2ME2 (lane 4 and 8), 25 μM pan PI3K inhibitor LY294002 (lane 3 and 7), these two compounds in combination (lane 5 and 9) or no treatment (lane 2 and 6), for 16 hours at 37°C and were exposed to 1% oxygen (lanes 2–9) or 20% oxygen (lane 1). β-actin was used as loading control. Both LY294002 and 2ME2 decreased hypoxic accumulation of HIF-1α, and together they more potent than either alone. (D) Role of PTEN in 2ME2-mediated regulation of HIF1α accumulation was evaluated using U373MG-Null and U373MG-PTEN cells with mutated p53. Cells were treated with inhibitor in similar conditions as described for U87MG Null and PTEN cells. 2ME2 alone had a more pronounced effect on the PTEN-reconstituted cells than PTEN-null cells, and the combination of 2ME2 with LY294002 was more potent than either drug alone. (E). LN229VIII cells were treated with 2ME2 (10μM or 25 μM), LY294002 (5μM or 25 μM) or in combination (5μM or 12.5 μM) followed by normoxia (20% oxygen) or hypoxia (1% oxygen) for 16 hrs.