*4.1. Reagents Preparation*

High-stability fucoxanthin (HS Fucoxanthin, HSFUCO, Fx) was obtained from Hi-Q Marine Biotech International Ltd. (Taipei, Taiwan) and dissolved in ddH2O [38].

#### *4.2. Cell Extraction and Treatment*

The extraction of primary rat valve interstitial cells (VIC) was carried out as shown in a previous study [42]. Briefly, after harvesting all the leaflets, pellet was centrifuged and incubated with collagenase II (Thermo) for 2 h to obtain VIC debris. Cells were then cultured in Dulbecco's Modified Eagle's Medium (DMEM/F12) (CASSION, Taichung City, Taiwan) combined with 100 units/mL of penicillin, 100 μg/mL of streptomycin (CORNING, Manassas, VA, USA), sodium bicarbonate (2.438 g/L; Bio-Shop, Burlington, ON, Canada), and 10% fetal bovine serum (FBS; CORNING, Manassas, VA, USA). To explore the protective effect of Fx against oxidative stress, VIC were pretreated with Fx for 24 h and then treated with H2O2 for 15 min.

#### *4.3. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Assay*

VIC cells were seeded in 96-well plates (3000 cells/well). The cells were pretreated with different doses of Fx (mg/mL) for 24 h and then with H2O2 for 15 min. After the treatment, we used the MTT assay (Abcam, Cambridge, MA, USA) for the analysis of cell viability. We added 1 mg/mL of MTT for 3 h until the crystal precipitation formed. Then added 100 μL/well of dimethyl sulfoxide (DMSO; ECHO Chemical Co. Ltd., Taipei, Taiwan) to dissolve the crystal formation. We used VERSA Max microplate reader (Molecular Devices, San Jose, CA, USA) to measure the optical density at 570 and 630 nm.

## *4.4. Cell Counting*

After the treatments, cell pellets were mixed with 0.4% trypan blue solution (Gibco, Grand Island, NY, USA). We used a hemocytometer (Hausser scientific company, Horsham, PA, USA) to calculate the cell number at 200× magnification.

## *4.5. PI Staining*

Propidium iodide (PI) solution (500 μg/mL) (Sigma-Aldrich, St. Louis, MO, USA) was dissolved with sterile ddH2O and stained with PI (1 μg/mL) solution for 1 h. We used microscopy (Olympus, Tokyo, Japan) to carry out fluorescence imaging at 200× magnification.

#### *4.6. ROS Density Measurement*

VIC cells were cultured in 6-well plates. We used 25 μM 2,7–dichlorofluorescin di-acetate (DCFDA, Cayman, Ann Arbor, MI, USA) staining for 30 min. Then, we used microscopy to capture the fluorescence image. We used the Image J software (Version 1.52t, NIH, Bethesda, MD, USA) to carry out ROS density quantification in single cells.

#### *4.7. Alizarin Red-S Staining Assay*

The calcification progression was assessed by Alizarin Red-S staining (Sciencell, Carlsbad, CA, USA). After treatment, the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 10 min and then stained with Alizarin Red-S for 30 min. We used a fluorescence microscope to image the stained area. We used the VERSA Max microplate reader to measure the absorbance at 405 nm.

#### *4.8. Protein Extraction and Western Blot*

Cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer with added protease and phosphatase inhibitors (Roche, Mannheim, Baden-Württemberg, Germany). Additionally, we quantified the cells with a bicinchoninic acid (BCA) assay, used sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a polyvinylidene fluoride (PVDF) membrane. Blocking with 5% bovine serum albumin (BSA) solution for 1 H was carried out. We used primary antibodies—poly (ADP-ribose) polymerase (PARP) (1:1000; Cell Signaling, Boston, MA, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:10,000; Proteintech, Rosemont, IL, USA), p-Akt (1:1000, Cell signaling), Akt (1:1000, Cell signaling), p44/42 MAPK (Erk1/2) (1:1000, Cell signaling), phospho-p44/42 MAPK (Erk1/2) (1:1000, Cell signaling), MMP-2 (1:1000, Abcam), BCL2

Associated X (Bax) (1:1000, Cell signaling), and B-cell lymphoma 2 (Bcl-2) (1:500, Santa Cruz, Santa Cruz, CA, USA)—cultured the mixture at 4 ◦C overnight, washed it three times, and then stained it with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000–10,000) for 2 h. The signal was captured by the eBlot Touch Imagertm (eBlot Photoelectric Technology, Shanghai, China). The band densities were determined using the Image J software program version 1.52 t (NIH, Bethesda, MD, USA). The expression level of these target proteins was analyzed in three individual experiments.

#### *4.9. In Vivo Animal Experiments*

With the help of a veterinarian, we recruited 26 heart disease-diagnosed dogs for the in vivo experiment. The dogs were treated with Fuco Pets HeartFight® (contained 60 mg/kg Fx) twice daily from Hi-Q Marine Biotech International Ltd. (Taipei, Taiwan) combined with medical treatments for 0.5 to 2 years. We used conventional echocardiography and standard Doppler examination to follow up the valve function. Esaote's MyLab ™ClassC® (Italy) equipped with a PA-122 probe cardio phased array (frequency range of 3–8 MHz) was used to obtain all the echocardiographic data.

The left atrium to aorta (LA/AO) ratio was measured using B-mode images acquired from a short axis five-chamber view of the right sternum wall.

## *4.10. Statistical Analysis*

The data are expressed as the mean ± standard deviation (SD). We used GraphPad Prism 8.0 for the analysis. Student's t-test was used for the comparisons between the two groups. One-way ANOVA tests were used to compare multiple groups, followed by Tukey's post hoc test. A *p*-value of less than 0.05 was considered significant. The p values are presented as \*, *p* < 0.05; \*\*, *p* < 0.01; \*\*\*, *p* < 0.001; or #, *p* < 0.05; ##, *p* < 0.01; and ###, *p* < 0.001.
