Figure 2
miR-99b controls RV replication by directly targeting mTOR. (A) HT29 cells were transfected with scrambled-miR or mimic/anti-miR-99b (40 nM) followed by western blot analysis using specific antibody against mTOR. Relative fold change was determined after normalization to GAPDH. (B) Lysed cell extracts either transfected with scrambled-miR or miR-99b (40 nM), were immunoprecipitated with anti-Ago2 antibody or isotype control i.e. rabbit IgG2a; following elution from beads. RNA was isolated and miR-99b bound mTOR mRNA expression was analyzed by qRT-PCR (left panel). mTOR and Ago2 expressions were analysed in the input lysates kept from each sample (right panel). Relative expression was determined after normalization to control cells. (C) Relative expression of miR-99b was determined in Ago2 immunoprecipitates by qRT-PCR. (D) Cells were mock infected or infected with RV followed by immunoblot analysis. Relative expression of mTOR, p-mTOR, and RV-VP6 was determined using specific antibodies. (E) HT29 cells were mock transfected or transfected with scrambled-miR or mimic/anti-miR-99b (40 nM) followed by RV-SA11 infection (6hpi); whole cell lysates were prepared, followed by immunoblotting analysis against mTOR, p-mTOR, and RV-VP6. Fold changes were measured after normalization with GAPDH. (F) HT29 cells were transfected with mimic/anti-miR-99b followed by RV-SA11 infection (6hpi) and viral titer was measured by plaque assay. All results are presented as the means and standard deviations from three independent experiments.
