FIG 4.

AMPK activity is required for 2 mM 2DG induced-reductions of rRNA transcription and H3K36me2 in rDNA promoter. (A) MCF-7 cells were cultured for 2 h in RPMI 1640 supplemented with 10% serum in the presence or absence of 2 mM 2DG with or without pretreatment of 10 μM compound C, an AMPK inhibitor, for 1 h, and the amounts of pre-rRNA were measured by qRT-PCR. The expression levels were normalized by Polr2 mRNA. The results were expressed as values relative to those without the treatments of compound C and 2DG. (B) Three days after transfection of control siRNA (control) or AMPKα 1/2 siRNA (siAMPK), MCF-7 cells were cultured 2 h in RPMI 1640 supplemented with 10% serum in the presence or absence of 2 mM 2DG. The amounts of pre-rRNA were measured by qRT-PCR. The results were expressed as in panel A. Knockdown of the AMPK subunit was confirmed by Western blotting shown in Fig. S3 in the supplemental material. (C) After cells were treated as for panel A, ChIP analyses were performed to detect H3K36me2, H3K36me3, and KDM2A in the rDNA promoter. The results were expressed as in Fig. 2B. All experiments were performed three times, and mean values with the standard deviations are indicated. *, P < 0.05.