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. 2014 Oct 24;22(5):815–825. doi: 10.1038/cdd.2014.176

Figure 5.

Figure 5

Co-regulation of Bcl-2 expression by Tra2β and miR-204. (a and b) After treatment with 10 nM control (ctrl) or TRA2β siRNA for 24 h, HCT116 cells were transfected with 25 nM control or pre-miR-204 was transfected for 24 h. The levels of mature miR-204 and BCL2 mRNA were measured by qPCR. *Significant changes compared with control siRNA-treated cells (P<0.05 by unpaired Student's t-test). (c) After treatment of HCT116 cells with 10 nM control or TRA2β siRNA for 24 h, they were transfected with the indicated concentrations of pre-miR-204 for 24 h. The levels of Tra2β and Bcl-2 were measured by western blotting. β-actin was used as a loading control. Bcl-2 signals were quantified by densitometry. Changes in relative intensities (Bcl-2/β-actin) are shown in the lower panel. Values are mean±S.D. from three independent experiments. *Significant changes compared with cells treated with control siRNA (P<0.05 by unpaired Student's t-test). (d) HCT116 cells were treated as in a and b. RIP assay was performed using an anti-Ago2 antibody or control IgG and lysates from cells transfected with the indicated oligonucleotides, and then BCL2 mRNA levels were measured by qPCR. *Significant changes compared with cells transfected with the YFP construct (P<0.05 by unpaired Student's t-test). (e) HCT116 cells were treated with 10 nM control or TRA2β siRNA for 24 h, and then transfected with 25 nM pre-miR-204 or pre-miR-448. BCL2α and BCL2β mRNA levels were measured by qPCR. *Significant change compared with control siRNA-treated cells (P<0.05 by unpaired Student's t-test). (f and g) After HCT116 cells were treated with 10 nM control or TRA2β siRNA for 24 h, they were transfected with 25 nM control or anti-miR-204 was transfected for 24 h. The levels of mature miR-204 and BCL2 mRNA were measured by qPCR. *Significant changes compared with control siRNA-treated cells (P<0.05 by unpaired Student's t-test). (h) HCT116 cells were treated as in f and g. The levels of Tra2β and Bcl-2 were measured by western blotting. β-actin was used as a loading control