(A) Northern blot analysis of miRNA expression in p53^WT and p53^-/- HCT116 cells with or without adriamycin treatment. Quantification of the miRNA signals under each condition, normalized to U6 snRNA expression, is shown on the right.
(B) Phylogenetic conservation of the genomic region near the transcription start site of the miR-34a primary transcript. VISTA (http://genome.lbl.gov/vista/index.shtml) was used to generate pairwise alignments between human and mouse and human and rat. The graph is a plot of nucleotide identity for a 100 base-pair sliding window centered at a given position. Position +1 of pri-miR-34a is the most 5′ transcription start site mapped. The magnified sequence shows the location and evolutionary conservation of the p53 binding site (in gray box). Mutations introduced into the promoter reporter construct (P1^mut in panel C) are shown in red.
(C) Activity of promoter constructs in p53^WT and p53^-/-cells. The arrow above construct P1 indicates the position of the transcription start site. Filled circles show the position of the p53 binding site. Error bars represent standard deviations from three independent transfections each measured in triplicate.
(D) Genotoxic stress activates the miR-34a promoter. The indicated promoter constructs were transfected into p53^WT or p53^-/- cells with or without subsequent exposure to adriamycin.

(A) p53^WT and p53^-/- cells were transfected with synthetic miR-34a or a control oligonucleotide and DNA content was monitored by flow cytometry. The fraction of apoptotic cells (sub-G1) is indicated.
(B) Cell death was monitored by Annexin V staining and flow cytometry. The right lower quandrant of each plot contains early apoptotic cells whereas the right upper quadrant contains late apoptotic cells. This experiment was repeated three independent times and similar results were obtained each time. Panels (A) and (B) come from two separate experiments.

Northern blot analysis of miR-34a expression in non-transformed pancreatic ductal epithelial cell lines (HPNE and HPDE, in bold) and in pancreatic cancer cell lines. Relative expression of miR-34a in each cell line, normalized to U6 snRNA expression, is shown below the blots. Dashes indicate undetectable miR-34a expression.

(A) Northern blot showing miR-34a expression in retrovirally-infected cell populations.
(B) Northern blot validation of selected expression changes observed by microarray analysis.
(C) Sites complementary to the miR-34a seed are enriched in the 3′ UTRs of downregulated transcripts. The graph shows the frequency of a site complementary to the miR-34a seed in the 3′ UTRs of the top 100 downregulated genes (2.364-fold or greater downregulation), the unchanged genes, and the genes upregulated by 2.364-fold or greater.