Fig. 3. Expression of exogenous mitochondrial-targeting DNMT1 impairs smooth muscle cell contractility.

a Vascular SMCs were treated with control (CL) or oligomycin (2 or 5 μmol/L) for 12 h and intracellular ATP content was assessed by a luminometer (n = 4). b–d Cells were embedded in collagen gel and were then treated with control (CL) or oligomycin (5 μmol/L) for 12 h in the absence (b) (n = 4) or presence of doxycycline (1 μg/mL) (c) or roscovitine (10 μmol/L) pre-treatment (12 h) (n = 5) (d). Cell contractions were determined by measuring the gel areas (n = 4). e–g Cells were transfected with empty vectors, no-MTS DNMT1, or MTS-DNMT1 and were then subjected to gel contraction assay in the absence (E) (n = 5) or presence of doxycycline (1 μg/mL) (f) or roscovitine (10 μmol/L) pre-treatment (12 h) (n = 6). h Single-cell contractility of SMCs was measured by traction force microscopy (TFM). Upper: Representative cell force images. Lower: Quantification of traction force with respect to the indicated treatments (n = 5). Scale bar: 25 μm. i Vascular SMCs were transfected with empty, no-MTS DNMT1, or MTS-DNMT1, and expressions of SMC contractile marker proteins were analyzed by western blot assay. Shown are representative blots (n = 3). j Semi-quantification of proteins in i. *P < 0.05, **P < 0.01, ***P < 0.005 by student’s t-test (b–d) and one-way ANOVA with Tukey’s post hoc analysis (a, e, f–h, j). Error bars show ± SEM.