Hoechst 33342

MEC cells were seeded in 6-well plates (5×10⁴ cells) with DMEM/High glucose supplemented as previously described and treated with Cephaeline (IC₅₀) in triplicates for 24 and 48h. After, cells were fixed with formaldehyde 4% for 15 min at room temperature. Blockage and cellular permeabilization were performed with 3% (w/v) bovine serum albumin (BSA) and 0.5% (v/v) Triton X-100 in PBS 1X for 1h. Anti- H3K9ac antibody (Cell Signaling Technology, Danvers, MA, USA) was diluted in (0.5% (v/v) Triton X-100 in PBS 1X and 1% (w/v) BSA) and incubated overnight. Subsequently, cells were washed and incubated with Alexa 488 secondary antibody (Cell Signaling Technology, Danvers, MA, USA) following with DNA staining using Hoechst 33342 (Cell Signaling Technology, Danvers, MA, USA). Ten fields of each slide were photographed and quantified. Images were taken using Nikon Eclipse Ti-S microscope and evaluated using Image J software (National Institute of Health, Bethesda, Maryland, USA).

Prior to excision, aortae were perfused serially via the left ventricle of the heart with: i) 0.5 mM EDTA in PBS; ii) 4% paraformaldehyde, 7.5% sucrose, 0.5 mM EDTA, in PBS; and iii) 0.5 mM EDTA in PBS, respectively. Dissected aortae were fixed in 4% paraformaldehyde for 30 minutes and then permeablized with 0.1% Triton X-100 in PBS at room temperature, followed by an overnight incubation (4°C) with a rabbit anti-β-Catenin antibody (Sigma #C2206) in 1:1000 diluted blocking solution (Dako-Agilent, K800621, Carpinteria, CA). Secondary anti-rabbit IgG antibody conjugated with Alexa Plus 555 (Thermo #14–387-071, Waltham, MA) at 1:1500 dilution was used for visualization of endothelial borders. Nuclei were counter stained with 1μg/mL Hoechst 33342 (Cell Signaling #4082). Vessels were mounted on a coverslip with ProLong anti-fade mounting medium (Thermo #P36962, Waltham, MA). Images were acquired with confocal microscope (Carl Zeiss) and ZEN 2012 software (Carl Zeiss). Since our cell culture work revealed no impact of sex, composite data from en face staining was typically sampled 4–8 times in aortae isolated from 4–5 mice of either sex. Representative staining in Figure 1 was derived from a male mouse and that in Figure 5 representes a female mouse.

Mitochondria in live cells were stained with either MitoTracker obtained from ThermoFisher (MitoTracker Red, M7512, Carlsbad, CA) or CytoPainter MitoRed from Abcam (#ab176832, Cambridge, MA) according to the manufacturer’s instructions. In brief, the cells were incubated with 1:1000 diluted MitoTracker/MitoRed in a growth medium for 15 minutes at 37°C incubator and followed by 1μg/mL Hoechst 33342 (Cell Signaling #4082) for 5 min at room temperature. Following the incubation, cells were washed in the growth medium twice and images were acquired.