Figure 4. miR-34 regulates a programme of cell cycle and DNA damage response genes.

a, Western blots were used to measure protein levels after miR-34 delivery for multiple candidate targets identified in the cell cycle overlapping gene set in Supplementary Fig. S6. Tub., tubulin. b, Reporter plasmids in which the luciferase coding sequence had been fused to the 3′ UTR of CDK4, CCNE2 or MET, as indicated, were transfected into HeLa cells in conjunction with either miR-34a (grey columns) or miR-124a (white columns) siRNAs. Luciferase activity was normalized relative to a simultaneously transfected Renilla expression plasmid. In each case 3′-UTR-Mut indicates the introduction of alterations into the seed complementary sites shown in Supplementary Fig. S8. Error bars indicate s.e.m. (n = 3). c, HCT116 Dicer^ex5 cells were transfected with siRNAs targeting CDK4, CCNE2 and MET, and cell cycle effects were analysed as described in Supplementary Fig. S6. The somewhat less efficacious arrest on transfection with CCNE2 siRNA could reflect a partly redundant function or less potent suppression of its mRNA.