Figure 3. miR-34 5′-end phosphorylation is ATM-dependent following DNA damage.

(a) A549 cells expressing psi-miR-34 (WT or MT) reporters were transfected with siRNA, exposed to 2 Gy and lysed at 12 h. Lysates were analysed for dual-luciferase activity. Renilla was normalized to Firefly and was expressed as the fold-suppression of MT/WT. Data are expressed as the fold change±s.d., relative to non-irradiated cells; n=2 independent experiments in triplicate. (b) ATM-deficient cells (GM16666) expressing wild-type ATM or a mutant (kinase dead) ATM were transfected with the psi-miR-34 (WT or MT) reporters. Cells were exposed to 2 Gy of IR and following a 4-h incubation, cells were analysed for miR-34 activity. Graphed is the average ±s.d. n=3 independent experiments. *P=0.035, two-tailed Student's t-test; **P=0.008, one-tailed Student's t-test. (c) 50 μg of total RNA extracted from A549 cells transfected with ATM siRNA or untransfected cells (from a) were untreated or treated with CIP and separated by denaturing PAGE. RNA was detected by northern blot with ³²P-labelled probes. tRNA stained with ethidium bromide was used for normalization.