(A) Immunofluorescence of Wt and Prog-Tg ECs using MRTFA antibody and DAPI (images are representative of n = 3 independent experiments). Scale bar: 10 μm. (B) Representative confocal average intensity projections of Z stacks from mouse aorta of Wt and Prog-Tg animals stained with DAPI and MRTFA and progerin antibodies. EC nuclei (arrowheads) lie on internal elastic membrane that displays blue autofluorescence. Arrowheads, MRTFA-positive ECs. Note that MRTFA accumulates at the nuclear periphery of progerin-positive (white arrowheads) but not progerin-negative (yellow arrowheads) ECs (n = 3 Wt and Prog-Tg littermate pairs). Scale bar: 10 μm. (C) Nos3 mRNA in Wt and Prog-Tg ECs after 24-hour treatment with 15 μM CCG-203971 MRTFA inhibitor or DMSO vehicle control. Values are normalized to Hprt and Nos3/Hprt values in Prog-Tg cells are shown relative to values in Wt cells after MRTFA-inhibitor and control treatment. Nos3/Hprt levels in Wt cells were arbitrary and set to 1 in both control and CCG-203971 conditions (n = 5 independent experiments). Nos3/Hprt levels in Prog-Tg cells relative to Wt are not significantly different (NS) after drug treatment, in contrast to control conditions (P < 0.01). (D) Chromatin immunoprecipitation using anti-MRTFA or goat IgG control. Precipitated DNA was amplified by qPCR with primers spanning Nos3 promoter or gene body (n = 3 independent experiments). **P < 0.01 by unpaired Student’s t test (C and D). Data presented as mean ± SEM. (E) Acta2 levels normalized to Hprt in fibroblasts after 3 days of coculture with Wt and Prog-Tg ECs either left untreated (left) or treated with 25 μM MRTFA inhibitor CCG-203971 (right). Values were subtracted from those obtained in untreated or CCG-203971–treated fibroblast single cultures (n = 6 Wt, n = 8 Prog-Tg, and n = 3 inhibitor-treated Prog-Tg samples). *P < 0.05 by Mann-Whitney U test. Data presented as median (middle line) with boxes encompassing 25th to 75th percentile, and whiskers, minimum to maximum values.