A Novel Anti-Apoptotic Role for Mir-290-295 Figure 5. Loss of the mir-295 cluster derepresses Casp 2 and Ei24 3’ UTRs and enhances sensitivity to DNA damaging agents. A. Luciferase reporters with full length versions of the Casp2 3’ UTR or Ei24 3’ UTR, as well as their seed mutant versions were assayed in 295 WT and 295 KO ES cells. B. Western blot of Casp2 in 295 WT and 295 KO ES cells. 50 nM miR-295, miR-467a, and miR-20a were transfected into 295 KO ES cells, and Casp2 protein expression was assayed 24 hours after the transfection. C. The percentage of cells with cleaved Casp3 in 295 WT and 295 KO ES cells after exposure to 5-Gy radiation (0 and 24 hours after radiation treatment). Cleaved Casp3 was assayed by flow cytometry, and was used to estimate apoptotic response. Apoptosis of 295 KO cells is shown in black bars, and that of 295 WT cells is shown in white bars. D. Cells were transfected with 50 nM siRNAs as shown, and difference in apoptotic response of 295 WT and 295 KO ES cells 24 hours after exposure to 5-Gy radiation was plotted. E,F. A similar series of experiments was performed on 295 WT and 295 KO cells after exposure to 100 nM doxorubicin. n$3 for all experiments. Results are shown as mean 6 S.E.M. (standard error of the mean). P-values were results of Mann-Whitney tests, and * denotes p#0.05, and ** denotes p # 0.01. doi:10.1371/journal.pgen.1002054.g005 PLoS Genetics | www.plosgenetics.org 7 May 2011 | Volume 7 | Issue 5 | e1002054