Figure 6.

Double knockdown of HuR and Transportin 2 prevented myogenesis. (A) C2C12 cells were transfected with the HuR siRNA duplex, targeting the HuR mRNA at the 111–131 sequence, the Trn2 siRNA duplex (Figure 3), or a combination of both, as well as a control siRNA duplex (Ctr) or transfection reagents only (−). Total cell extracts were prepared 24 h after transfection, and Western blotting was performed using antibodies to Trn2, HuR, and β-actin to demonstrate the degree of knockdown of each (HuR and Trn2) protein. (B) Western blot analysis of 20 μg of total cell extracts harvested on day 2 of differentiation was performed, and the membrane was probed with antibodies to myoglobin and β-actin. (C) C2C12 cells that were transfected with siRNA (transfection reagents only [mock] Trn2, HuR, Trn2+HuR, and control) were fixed on day 3 of differentiation. Immunofluorescence staining with anti-myoglobin was performed to determine the differentiation status of the transfected cells. DAPI, myoglobin, and merged (DAPI+myoglobin) images from a single representative field of view are shown. (D) The fusion index of myotubes was calculated to assess the percentage of differentiation in C2C12 cells under each transfection condition. The quantitative analysis was performed as described in Figure 1. Error bars, SD of two independent experiments.