Fig. 2. VPA inhibits p-MLC-Ser¹⁹ by reducing RhoA expression and the levels of active RhoA-GTP. (A) VSMCs were treated with various doses of VPA (0, 1, 2, or 4 mM) for 24 hours, followed by stimulation with 1 μM PE for 10 minutes, and then levels of RhoA and actin expression were detected using western blotting. (B) VSMCs were treated with 2 mM VPA for various duration (0, 3, 8, 24 hours), followed by stimulation with 1 μM PE for 10 minutes, and then levels of RhoA and actin expression were detected using western blotting. (C) VSMCs were treated with various doses of VPA (0, 1, 2, or 4 mM) for 24 hours, followed by stimulation with 1 μM PE for 10 minutes, and then levels of RhoA mRNA expression relative to GAPDH mRNA expression were detected using qRT-PCR. (D) VSMCs were treated with 2 mM VPA for various duration (0, 3, 8, 24 hours), followed by stimulation with 1 μM PE for 10 minutes, and then levels of RhoA mRNA expression relative to GAPDH mRNA expression were detected using qRT-PCR. (E) After VSMCs were treated with various doses of VPA (0, 1, 2, or 4 mM) for 24 hours, followed by stimulation with 1 μM PE for 10 minutes, RhoA activity assay was performed and levels of active RhoA-GTP, RhoA, and actin expression were determined using western blotting. (F) After VSMCs were transfected with the CA-RhoA mutant gene or empty vector and treated with 2 mM VPA for 24 hours, followed by stimulation with 1 μM PE for 10 minutes, levels of RhoA, myc, and actin expression were detected using western blotting. (G) After VSMCs were treated as described above, levels of p-MLC-Ser¹⁹, MLC, and actin expression were detected using western blotting. All experiments were performed independently at least four times, and the blots shown are representative of these experiments (n = 4). Bar graphs depict the mean above/below the control (± standard deviation).

VPA = valproic acid, p-MLC-Ser¹⁹ = phosphorylation of the myosin light chain at Ser19, RhoA = Ras homolog family member A, GTP = guanosine-5'-triphosphate, VSMC = vascular smooth muscle cell, PE = phenylephrine, GAPDH = glyceraldehyde-3-phosphate dehydrogenase, qRT-PCR = quantitative real-time reverse transcription-polymerase chain reaction, CA = constitutively active, MLC = myosin light chain.

Differences were considered statistically significant at ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001.