Figure 3

Analysis of ms1 promoter function in myogenic cell lines. (A) Subconfluent NIH3T3 fibroblasts and C2C12 myoblasts were transiently transfected with a ms1 promoter reporter construct spanning from -1585 relative to transcription start site and extending to nucleotide +60. Promoter fragment was cloned into the pGL3-Basic luciferase vector. Activity relative to pGL3-B alone was determined in each cell line 48 hours later, at which point the cells were still subconfluent. The relative activity of the promoter fragment (vs pGL3-B) in each cell line was then determined with relative activity in C2C12 assigned arbitrary value of one (B) Vectors (+:0.3 μg, ++:0.6 μg) expressing Mef2D, myogenin and MyoD were co-transfected in combination with the ms1 prompter reporter construct (P-1585/+60) into C2C12 myoblasts as described in (A). Luciferase activity in cells transfected with pcDNA and ms1 promoter reporter (P-1585/+60) was arbitrarily set at 1 fold activation. (C) Subconfluent H9c2 myoblasts were transiently transfected with the MRF over-expression vectors (+:0.5 μg, ++:1 μg). 48 hours post transfection, at which point the cells were 80% confluent, total RNA was isolated, reverse transcribed and the expression levels of TATA binding protein (TBP) and ms1 were determined by real time PCR. Ms1 expression in each sample was normalised to that of TBP. Statistically significant differences are indicated by *P < 0.05.