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. Author manuscript; available in PMC: 2014 Jul 1.
Published in final edited form as: Clin Cancer Res. 2013 May 21;19(13):3429–3438. doi: 10.1158/1078-0432.CCR-12-3794

Figure 3. miR-452 directly downregulates multiple stemness regulators.

Figure 3

A. Predicted miR-452 target sequences in the Bmi-1–3′UTR, LEF1–3′UTR and TCF4–3′UTR and mutants containing two mutated nucleotides in 3′UTR of these transcripts. B. Western blotting analysis of the expressions of Bmi-1, LEF1 and TCF4 in the indicated cells. α-Tubulin was used as a loading control. C. Luciferase activities of reporters containing the 3′UTRs or mutated-3′UTRs of Bmi-1, LEF1 and TCF4 in the indicated cells transfected with negative control (NC) or miR-452 mimic. D. Upper panel: miRNP IP assay showing the association of miR-452 with Bmi-1, LEF1 and TCF4. GAPDH was used as a negative control and 5S rRNA was used as a control for overall expression levels. Lower panel: Western blotting analysis of HA-Ago1. E. Upper panel: Restoration of Bmi-1, LEF1 and TCF4 expression partially, but significantly, rescued the proportion of SP cells in miR-452-transduced cells. Lower panel: Western blotting analysis of Bmi-1, LEF1 and TCF4 protein expression in the indicated cells. α-Tubulin was used as a loading control. Error bars represent the mean ± SD of three independent experiments. * P, < 0.05.