Figure 4. miR-34 activation, phosphorylation and localization.

(a) A549 cells expressing psi-miR-34 (WT or MT) reporters were transfected with siRNA, exposed to 2 Gy and lysed at the indicated time. Lysates were analysed for luciferase activity and miR-34 expression by RT–qPCR. Renilla was normalized to Firefly and was expressed as the fold-suppression of MT/WT. MiR-34 expression was normalized to U6. Data are expressed as the fold change±s.d., relative to non-irradiated cells; n=4 independent experiments. (b) Untreated or irradiated (6 Gy) A549 cells were lysed and ATM, Clp1 or Vimentin were immunoprecipitated. Immunoprecipitates (or T4 PNK) were incubated with 3′-biotinylated RNAs and gamma-32P ATP (6,000 Ci mmol⁻¹). RNA was selected using Avidin-D Agarose. RNA was eluted by phenol extraction, precipitated and analysed by 10% Urea-PAGE. Ethidium bromide-stained gel is shown as a loading control. (c) A549 cells were untreated or exposed to 6 Gy. Following a 3-h incubation, cells were lysed and 50 μg of lysate was incubated with 1 μg of ATM, IgG, Clp1 or Vimentin antibody (as indicated). Antibodies were captured with Protein A/G agarose and protein was eluted with SDS–PAGE sample buffer. Samples were separated on a 4–20% gradient gel and transferred to PVDF. ATM was probed for using anti-ATM antibody. The input control is 1/100th of the lysate used to IP. (d) Total RNA was isolated from nuclear and cytoplasmic fractions at different time points post irradiation with 4 Gy in A549 cells. miR-34 miRNA was measured and data are expressed as the average fold change±s.d. after normalization to miR-17. Samples were run in triplicate and the experiment was repeated four separate times. P-value is based on a two-tailed, two-sided t-test. **P=0.045.