TNFα stabilizes myocardin mRNA which upregulates myocardin expression and activity in differentiated VSMCs.

<p>(<b>A</b>) VSMCs were serum-starved for 24 h and transcription was blocked using α-amanitin (50 µM). Cells were then treated with TNFα (50 ng/ml) for 1, 3, 6 and 9 h. Graph shows the effect of TNFα on myocardin mRNA degradation. (<b>B</b>) VSMCs containing the T-REx system for myocardin over-expression were treated with Dox. Western blot analysis shows the effect of Dox on myocardin and contractile protein expression levels. (<b>C</b>) RT-qPCR analysis showing the effect of myocardin over-expression on MRTF-A and B. (<b>D</b>) Light microscope images showing the effect of Dox on VSMC morphologies. (<b>E,F,G</b>) VSMCs were serum-starved for 24 h and treated with TNFα (50 ng/ml) and Dox for 24 h. Representative western blots and RT-qPCR analysis show the effect of TNFα on myocardin and contractile marker protein and mRNA expression levels compared with those control without Dox (C). (<b>H</b>) VSMCs without the T-REx system to over-express myocardin were treated with TNFα and Dox to determine non-specific effects of Dox on myocardin. RT-qPCR analysis shows the effect of TNFα and Dox on myocardin mRNA expression levels compared with the control (C). (<b>I</b>) Primary rat aortic VSMCs (passage 2) were serum-starved for 24 h and treated with TNFα (50 ng/ml) for 24 h. Representative western blots show the effect of TNFα on myocardin and contractile marker protein expression levels. (<b>J,K</b>) Western blot and RT-qPCR analyses showing the concentration- and time-dependent effects of TNFα on myocardin and contractile marker expression in VSMCs treated with Dox. qPCR results were normalized using GAPDH (n = 7, *p<0.05).