Figure 4.

Bim repression by Hoxb8 is mediated at the Bim 3′UTR with possible regulation by miRNAs. (a) Reporter activity generated by different segments of the Bim promoter and 3′UTR. 293T cells stably expressing GFP or Hoxb8 were transiently transfected with luciferase reporter constructs containing either the 3.6 kB Bim promoter upstream of Exon 1, Intron 1 or the 3′UTR. Cells were co-transfected with renilla luciferase to control for transfection efficiency. Luciferase levels are expressed relative to the GFP-expressing 293T cells. Results are mean±S.E.M. of three independent experiments. I: Intron, E: Exon, L: Luciferase. Structure of murine Bim gene is shown (b) Wild-type Hoxb8-FDM cells were cultured in IL-3 and in the presence (+) or 4-day absence of 4-OHT (−). The left panel shows the fold change in expression in response to 4-OHT for the most affected miRNAs. For each miRNA, the change in Ct value in response to 4-OHT is shown relative to the average Ct of all the six samples. This scheme allows visualisation of the degree of change for all the levels of expression. The heat map on the right shows the actual Ct values, averaged across the three biological replicates for each treatment. Highest expression is represented as red and the lowest expression as blue. Red arrows indicate guide strands from the miRNAs of the miR-17∼92 cluster of miRNAs. (c) Table of miRNAs with significant fold changes as observed from (b) that are members of the miR-17∼92 cluster on chromosome 13 and related genes on chromosomes 7 and X. P-values of fold changes were calculated using a Student's t-test (independent, two-tailed). Indicated are both the -5p and -3p strands of each miRNA corresponding to the fold change observed for each in the qPCR array. This includes miRNAs previously known as the star forms