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. 2003 Apr;23(7):2425–2437. doi: 10.1128/MCB.23.7.2425-2437.2003

FIG. 4.

FIG. 4.

Myocardin-induced transactivation of multiple SMC-specific transcriptional regulatory elements in COS-7 and undifferentiated mouse ES cells. (A) cis-acting mechanisms regulating myocardin-induced transactivation of the SM22α promoter in COS-7 cells. The nucleotide sequence of SME-4 (bp −171 to −136) and SME-4 deletion mutants CArG (bp −150 to −141), 5′CArG (bp −171 to −141), and 3′CArG (bp −151 to −136) are shown. COS-7 cells were cotransfected with the pcDNA-Myocardin expression plasmid, the phRL-TK(-Int) reference plasmid, and the indicated reporter plasmid. Luciferase activities were measured 48 h posttransfection. Myocardin-induced transcriptional activation of luciferase reporter plasmids placed under the transcriptional control of the 441-bp mouse SM22α promoter (−441.luc), the SM22α promoter containing mutations that abolish SRF binding (−441μCArG.luc), the 90-bp SM22α promoter linked to four copies of SME-4 (SME4×4.luc), the 90-bp SM22α promoter linked to four copies of the CArG oligonucleotide (CarGx4.luc), the 90-bp SM22α promoter linked to four copies of the 5′CArG oligonucleotide (5′CarGx4.luc), and the 90-bp SM22α promoter linked to four copies of the 3′CArG oligonucleotide (3′CarGx4.luc). Luciferase activity is reported as induction of luciferase activity observed when each reporter plasmid was cotransfected with pcDNA-Myocardin relative to the activity observed with pcDNA3. Results are expressed as the mean ± SEM. (B) Myocardin-induced transactivation of multiple SMC-specific transcriptional regulatory elements in mouse ES cells. Undifferentiated mouse ES cells were cotransfected with the pcDNA-Myocardin expression plasmid, the phRL-TK(-Int) reference plasmid, and the indicated reporter plasmid. Myocardin-induced transcriptional activation of luciferase reporter plasmids placed under the transcriptional control of the 441-bp mouse SM22α promoter (−441.luc), the SM-α-actin promoter and intragenic enhancer (pPIAct-luc), and the SM-MyHC promoter and intragenic enhancer (pPIMyo-luc) is reported as the induction in luciferase activity observed when cotransfected with pcDNA-Myocardin relative to the activity observed with pcDNA3. Results are expressed as the mean ± SEM. All transfections were repeated at least three times to ensure reproducibility.