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. 2019 Mar 12;8:e43511. doi: 10.7554/eLife.43511

Figure 3. Luciferase assays to assess putative miR-34a binding sites in the 3’UTR of CD44.

Figure 3.

DU145 cells were transfected with either a luciferase reporter with a fragment of the 3’UTR of CD44 with both putative miR-34a binding sites left intact (wt) or mutated (M1M2). Cells were also co-transfected with miR-34a oligos (34a) or control oligos (NC). (A) Luciferase (Luc) activity was normalized to the NC condition for both luciferase reporters for each biological repeat such that NC conditions were assigned a value of 1. Means reported and error bars represent s.e.m. from 16 independent biological repeats. Wilcoxon-Mann-Whitney test comparing Luc values from wt +34 a vs M1M2 + 34 a; U = 104, uncorrected p=0.381, Bonferroni adjusted significance threshold = 0.0167; (Bonferroni corrected p>0.99). One-sample Wilcoxon signed-rank test comparing Luc values from wt +34 a to a constant of 1 (wt +NC); z = 0.517, uncorrected p=0.632; (Bonferroni corrected p>0.99). One-sample Wilcoxon signed-rank comparing Luc values from M1M2 + 34 a to a constant of 1 (M1M2 + NC); z = 0.310, uncorrected p=0.782; (Bonferroni corrected p>0.99). (B) This is the same data as in part (A), but Luc activity was not normalized. Luc values are presented relative to wt +NC. Box and whisker plot (y-axis is log10 scale) with median represented as the line through the box and whiskers representing values within 1.5 IQR of the first and third quartile. Kruskal-Wallis test on all four groups: H(3) = 11.1, p=0.011. Wilcoxon-Mann-Whitney comparison between luciferase reporter (wt or M1M2): U = 266, uncorrected p=7.67×10−4, Bonferroni corrected p=1.53×10−3, Cliff’s d = 0.48, 95% CI [0.19, 0.69]. Wilcoxon-Mann-Whitney comparison between oligo (NC or 34a): U = 497, uncorrected p=0.847, Bonferroni corrected p>0.99, Cliff’s d = 0.03, 95% CI [−0.25, 0.30]. Wilcoxon-Mann-Whitney comparison between wt +NC and wt +M1M2: U = 119, uncorrected p=0.752, Bonferroni corrected p>0.99, Cliff’s d = 0.07, 95% CI [−0.32, 0.44]. Additional details for this experiment can be found at https://osf.io/vzb82/.