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p53 induces all miR-34 genes in zebrafish but with different kinetics. (A) Quantitative PCR (qPCR) analysis of miR-34 genes (miR-34a, miR-34b, miR-34c) as well as p53 target genes p21 and cycG1 after 1, 2, 4 and 5 hours of treatments at 24 hpf with 0.1% DMSO or 1μM camptothecin (CPT), which induces DNA damage and p53 activation. (B) qPCR analysis of miR-34 genes as well as p53 target genes p21 and cycG1 after 1 and 3 hours of treatments at 48 hpf with 0.1% DMSO or 1 μM CPT. (A, B) The qPCR experiment was run with 4 biological replicates and 2 technical replicates. (C) Whole mount in situ hybridization analysis of miR-34a expression pattern after 4-hour treatment with DMSO or CPT in wild-type zebrafish embryos. Representative images of 40 embryos stained per condition are shown. (D) qPCR for p53 target genes (p53, p21) and miR-34 genes in wild-type and p53 null mutants after a 4-hour treatment at 24 hpf with either 0.1% DMSO or 1μM CPT. The qPCR experiment was run with 7 or 8 biological replicates and 2 technical replicates. Fold changes for each gene are indicated on the log10-scaled y-axis relative to control. Standard errors are used for error bars. Significantly different genes in (A, B, D) are indicated by ‘***’ (P-value < 0.001), ‘**’ (P-value < 0.01) and ‘*’ (P-value < 0.05). (E) Positions and alignments of the p53 transcription factor motif in the 20-kb regions around the miR-34a and miR-34b/c genes performed using the MAST software. The alignment figure position above or below the genomic sequence line indicates the strand (+ or -, respectively). The P-values for the motif matches are indicated on the alignment inserts.
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