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Figure S3. MKK6EE rescues the block to C2C12 cell differentiation imposed by RNAi to Cdo. C2C12 cells were cotransfected with control or Cdo RNAi expression vectors, control or MKK6EE expression vectors, and pQ-lacZ, a vector that drives the expression of nuclear-localized β-galactosidase, to mark transfectants. 48 h after transfection, the cultures were transferred to DM, and, 48 h later, the cells were double stained for β-galactosidase activity and for MHC expression as described previously (Kang, J.-S., M.-J. Yi, W. Zhang, J.L. Feinleib, F. Cole, and R.S. Krauss. 2004. J. Cell Biol. 167:493-504). (left) Photomicrographs of cultures (blue color indicates β-galactosidase activity, and brown color indicates immunostaining for MHC). (right) Quantification of C2C12 cell differentiation. Approximately 15% of double control vector transfectants (purple bars) are MHC^-, ∼48% are MHC⁺ and mononucleated, and ∼37% are MHC⁺ and multinucleated. The expression of Cdo RNAi (pink bars) increases the percentage of MHC^- cells to >45% and decreases the percentage of multinucleated MHC⁺ cells to - cells, MHC⁺ and mononucleated cells, and MHC⁺ and multinucleated cells to values similar to those found in control transfectants. The expression of MKK6EE alone (yellow bars) increased the percentage of multinucleated MHC⁺ cells, but its effects were less impressive than its effects on cells expressing Cdo RNAi. Values represent means of triplicate determinations ± SD (error bars). The experiment was repeated three times with similar results. The transfection efficiencies for these experiments were ∼10%, a value chosen to minimize fusion of independent β-galactosidase⁺ transfectants (Kang, J.-S., M.-J. Yi, W. Zhang, J.L. Feinleib, F. Cole, and R.S. Krauss. 2004. J. Cell Biol. 167:493-504).