Figure 6. AMPK-mediated Phosphorylation of EZH2 at T311 Attenuates PRC2 Oncogenic Function.
(A) EZH2-depleted OVCA5 cells stably reintroducing control, WT, T311A, R308L, T311E and S21D EZH2 were quantified for mRNA level expression of EZH2 target genes. mRNA levels of HOXA11, OLIG2, SXO17 and GATA6 were measured by qRT–PCR, and are normalized to GAPDH expression. Data are means ± SEM (n=3).
(B–C) EZH2-depleted OVCAR5 cells stably expressing GFP (as a negative control) or the indicated HA-EZH2 constructs were infected with shGFP or shEZH2 lentiviral constructs. Infected cells were selected with 1 μg/ml puromycin for 72 hours to eliminate the non-infected cells. These cells were subjected to clonogenic survival assays for 10 days. Crystal violet was used to stain the formed colonies (B). The colony numbers were calculated as mean ± SD (n=3), * P < 0.05, Student’s t test (C).
(D–E) OVCAR5 cells generated in (B) were seeded (10,000 cells per well) in 0.5% low-melting-point agarose in DMEM with 10% FBS, layered onto 0.8% agarose in DMEM with 10% FBS. The plates were cultured for 21 days whereupon the colonies >50 μm were counted under a light microscope (D). The colony numbers were plotted as mean ± SD (n=3), * P < 0.05, Student’s t test (E).
(F–G) The tumor size (F) and growth curves (G) for the xenograft experiments using shEZH2-OVCAR5 cells expressing GFP (as a negative control) or the indicated EZH2 construct. In each flank of six nude mice, 1×106 cells were injected subcutaneously. The visible tumors were measured at the indicated days. Error bars represent ±SEM, n=6.
(H–I) EZH2-depleted OVCAR5 cells stably expressing WT or R308L-EZH2 constructs were subjected to clonogenic survival assays for 14 days. 100 μM A769662 was added immediately after plating cells as indicated. Crystal violet was used to stain the formed colonies (H). The colony numbers were calculated as mean ± SD (n=3) (I).
(J–K) EZH2-depleted OVCAR5 cells stably expressing WT or R308L-EZH2 constructs were seeded (10,000 cells per well) in 0.5% low-melting-point agarose in DMEM with 10% FBS, layered onto 0.8% agarose in DMEM with 10% FBS. The plates were cultured for 21 days whereupon the colonies >50 μm were counted under a light microscope. 100 μM A769662 was added immediately after plating cells as indicated (J). The colony numbers were calculated as mean ± SD (n=3) (K).
See also Figure S6.