Hoechst

MSCs treated with H₂O₂ and/or Gel were cultured on cell slide in 24-well plate bottom with DMEM medium containing 10% FBS. MSCs on cell slide were fixed in 4% paraformaldehyde for at least 30 min at room temperature (RT) and then washed with PBS for 10 min at RT. Permeabilization and blocking were carried out simultaneously in PBS containing 15% FBS, 2% BSA, and 0.1% saponin (all Sigma-Aldrich, USA) for 30 min at RT. Cells were rinsed with PBS and incubated overnight at 4°C with primary antibodies diluted 1:100 in PBS containing 15% FBS and 2% BSA. Primary antibodies were Rabbit anti-MYOG (Proteintech, 18943-1-AP) and Rabbit anti-MYOD1 (Abcam, ab1835). Stained cells were then washed three times for 10 min each with PBS followed by secondary antibodies diluted 1:100 in PBS. Hoechst (Vector Laboratories, USA) for nuclear/DNA labeling was used at a 1:1000 dilution in PBS. Cells were washed three times for 10 min each and quantified in ProLong Gold (Vector Laboratories, USA) overnight at 4°C.

While sacrificing mice that were fed a WD for 7 months (see 2.2.), hearts and aortas were embedded in optimal cutting temperature (OCT) compound and snap-frozen for further analysis. Frozen sections of 10 μm thickness were stained with oil-red-O, and serial 5 μm cryo-sections of the aortic sinus area were used for immunostaining as previously described [19] . For immunostaining, sections were fixed in cold acetone for 10 min, then washed with PBS for 2 times, blocked in background buster (Innovex) at 37 °C for 1 h, then incubated with rat anti-mouse CD106 (Millipore) for identifying VCAM-1 at 4 °C for overnight. The next morning, sections were washed with PBS for 3 times, then incubated with Alexa Fluor 488 goat-anti rat IgG (Invitrogen) and at 37 °C for 1 h. Slides were washed, and cell nuclei were counterstained with Hoechst (Vector Labs, Burlingame CA). Images were captured using Olympus IX81 microscope. Quantification of images was performed as described before [19] . For quantification of VCAM-1 expression in the lesion, multi-channel images were split first and the density (green component only) of VCAM-1 was measured using Image J software. Blind analysis was performed for all images. At least two sections from each mouse were analyzed and all the experimental mice (n ≥ 19) were used for analysis.

The effect of magnolol on the nuclear translocation of p65 was evaluated by immunofluorescence staining. Briefly, BMMCs treated with RANKL in the absence or presence of 20 µg/ml magnolol were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 in PBS for 5 min. The cells were washed with 0.1% BSA-PBS and incubated with an anti-p65 monoclonal antibody overnight at 4°C, followed by a biotinylated goat anti-mouse IgG and fluorescein-conjugated streptavidin (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature and Hoechst (Vector Laboratories) was used for counterstaining for 2 days. The localization of p65 was visual-ized by immunofluorescence analysis (×400 magnification) and the percentage of nuclei fluorescence intensity of p65 in 3 random fields were measured in each group with ImageJ software.