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. 2007 Aug;18(8):2864–2872. doi: 10.1091/mbc.E06-12-1069

Figure 3.

Figure 3.

Inhibition of ERF gene expression enhances utrophin mRNA levels and utrophin-A promoter activity in C2C12 cells. (Ai) Semiquantitative RT-PCR of ERF, utrophin, and GAPDH transcript levels in murine C2C12 cells after treatment with either 100 pmol (left lane) of an unrelated, scrambled control egg oligomer, or 25 nmol each of four siRNA complementary to ERF. ERF siRNA oligomers caused a specific decrease in ERF transcript to ∼2% and an ∼1.4-fold increase in utrophin mRNA after adjustment to GAPDH levels. (ii) ERF knockdown causes an increase in utrophin promoter activity. The pPUBF utrophin promoter-luciferase construct was transfected into C2C12 cells 24 h after either egg or ERF siRNA oligomer transfection, and luciferase activity was assayed after an additional 24-h incubation. An approximate twofold elevation of utrophin promoter activity in C2C12 cells was observed with the ERF oligomer mix (1.99 ± 0.24) in comparison with the egg scrambled control. Luciferase values are the means of four separate experiments performed in triplicate (n = 12). Error bars are SEM. (iii) Semiquantitative RT-PCR of MyoD, myogenin, and GAPDH transcript levels in murine C2C12 cells after treatment with either 25 nmol each of four siRNA complementary to ERF (left lanes, 1–3) or 100 pmol (right lanes, 1–3) of an unrelated, scrambled control egg oligomer and assayed after 24 h. The − marked lane shows a negative control for RT-PCR where reaction was performed without template. No significant changes in MyoD or myogenin transcript levels were noted. Gels show results of three separate experiments. (B) Chromatin immunoprecipitation analysis of the utrophin-A promoter with ERF antibodies in C2C12 cells, demonstrating changes in ERF occupancy of the utrophin-A promoter upon treatment with MEK inhibitor U0126 and HRG treatment for 15 min. (i) RT-PCR of utrophin-A promoter from ERF-precipitated chromatin from C2C12 cells. (ii) Quantification of changes in ERF occupancy compared with the untreated ERF controls shows increased (1.63-fold) and decreased (0.64-fold) ERF levels with MEK inhibitor U0126 and HRG, respectively. Controls incubated without antibodies are shown below.