Figure 2. EDD is a key effector for miRNA silencing.

A. In an ES cell reporter cell line (1A2E8), Rn-Luc activity is repressed by λN22HA-Ago2. Ff-Luc serves as an internal control. B. EDD is required for λN22HA-Ago2-mediated translation repression of Rn-Luc reporter. Shown are relative ratios Rn/Ff-luc activity and mRNA abundance (n=3). Rn-Luc activity was significantly increased in the reporter cells expressing different EDD shRNAs while no obvious change was seen for Rn-Luc mRNA. C. Western blot showing that EDD was significantly downregulated in EDD-shRNA-expressing 1A2E8 cells. D–E. miRNA silencing is significantly compromised in EDD knockdown STO cells. STO fibroblasts were transiently transfected with EDD shRNA-4 and dual luciferase reporters and effectors. Both mir-CXCR and mir-30 reporter assays demonstrated a defect in miRNA silencing in EDD knockdown cells. F. An inducible shRNA expression system in HeLa cells. HeLa cells are first stably transfected with a PiggyBac (PB) vector expressing Tet repressor (TetR) under the control of CAG promoter. An inducible RNAi PB transposon is subsequently introduced to express EDD shRNA. The shRNA is under the control of a chimeric human H1 and TetO promoter. The integration of H1/TO-shRNA is selected with puromycin resistance conferred by PGK-TetR-IRES-Puro-bPA in the same PB transposon. In the absence of doxcycline (Dox), TetR suppresses shRNA expression. Upon Dox treatment, shRNA is induced. G. miRNA silencing defects in Dox-induced EDD knockdown HeLa cells. Endogenous miRNA targets p27Kip1 and HMGA2 were upregulated upon EDD depletion in HeLa cells. Due to the sensitivity of HMGA2 antibody and the expression level of HMGA2 in HeLa cells, endogenous HMGA2 was only detected 2 days after EDD knockdown. H. In EDD knockdown HeLa cells, HMGA2 mRNA was upregulated. RT-PCR and RT-qPCR showed an increased level of HMGA2 message 2 days after Dox induction. HMGA2 mRNA abundance was normalized to actin in each sample. For the left panel, 32 cycles were used for HMGA2 and 21 cycles were used for actin. I. The let-7 HMGA2-3′UTR reporter assay in HeLa cells confirmed that increased levels of HMGA2 protein and HMGA2 mRNA were due to a defect in miRNA-silencing. In the absence or presence of Dox, inducible EDD knockdown HeLa cells were transfected with Rn-HMGA2-3′UTR-WT or Rn-HMGA2-3′UTR-m7 (in which 7 let-7 binding sites had been mutated). Ff luciferase was used as an internal control. The let-7 mediated repression was measured by comparing normalized expression levels for Rn-HMGA2-3′UTR-WT versus Rn-HMGA2-3′UTR-m7. The result revealed a defect of let-7 mediated HMGA2 silencing in EDD-depleted HeLa cells. All results are shown as means ± SEM from 3–6 independent transfections and subjected to two-tailed t-tests. * p < 0.05, ** p < 0.01, *** p < 0.0001.