Figure 2

Sumoylation-deficient mutant of MEF2C promotes myogenic conversion more efficiently. (A) MEF2C-WT- or MEF2C-K391R-expressing plasmids were co-transfected with MEF2×3-luciferase reporter, pRL-tk reporter, and GFP-SUMO1 or empty vector plasmids into HeLa cells. Firefly luciferase activities were measured and normalized for transfection efficiency by using Renilla luciferase activities. (B) MEF2×3-luciferase reporter, pRL-tk reporter, and MKK6-DD plasmids were co-transfected with GFP-SUMO1, SENP2, or empty vector plasmids into C2C12 cells. The cells were cultured in differentiation medium for 2 days. Firefly luciferase activities were measured and normalized for transfection efficiency by using Renilla luciferase activities. (C) Vector, MEF2C-WT-, MEF2C-K391R-, or GFP-SUMO1-expressing constructs were co-transfected with Myc-MyoD into 10T1/2 cells. The cells were cultured in differentiation medium for 5 days, fixed, and stained with DAPI (blue) and an anti-myosin heavy chain (MHC) monoclonal antibody (red). (D) MHC-positive cells were scored by random selection of 20 optical fields of cells in (C). The results of two independent experiments were averaged with the standard deviation indicated. P value was calculated using student t test. Cell lysates were resolved by SDS-PAGE and blotted with anti-MEF2C or Myc.