*4.7. Western Blot Analysis*

B16-F10 cells (2 × 10<sup>6</sup> cells per well) were seeded into a 10 cm dish for 24 h, and then cells were treated with di fferent concentrations of PSS (12.5, 25, 50, 100 μg/mL) for 24 h. The medium was removed, and the cells were washed with PBS three times. Cells were then lysed in 200 μL of lysis bu ffer on ice. The total protein was determined using the Bicinchoninic Acid (BCA) Kit (Solarbio, Beijing, China). Equal amounts of protein in the cell extracts were fractionated by 10% SDS-PAGE, and then electrotransferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with TBST (20 mM Tris-bu ffered saline and 0.1% Tween) containing 5% nonfat dry milk for 1 h at room temperature, the membranes were incubated for 2 h with monoclonal antibodies, such as anti-MMP-9, anti-MMP-2, anti-E-cadherin, anti-Vimentin, anti-ERK 1/2, anti-p-ERK 1/2, anti-AKT, anti-p-AKT (Ser473), anti-p38, anti-p-p38, anti-p-p38, anti-NF-κB, anti-p-NF-κB, and anti-β-actin, which were purchased from Cell Signaling Technology. The membranes were then washed three times and incubated with HRP-conjugated secondary antibodies (Abcam, Cambridge, Massachusetts, USA). The proteins were then detected using chemiluminescence agents (Amersham ECL, GE Healthcare, Buckinghamshire, UK).

### *4.8. In Situ Zymography Localization of Matrix Metalloproteinase 2 and Matrix Metalloproteinase 2 Activity*

MMP-2/9 activity was tested on the slides of cells using the GENMED Kit (Genmed Scientifics Inc., Wilmington, DE, USA) according to the manufacturer's instructions (GENMED80062.4/80062.6, GENMED). B16-F10 were seeded in 12-well culture plates with a glass slide until they reached 50% confluence. After washing with PBS, fresh serum-free culture media was added to the plate in the presence or absence of serial PSS treatments, at concentrations ranging from 25–100 μg/mL. After 24 h, the glass slides were carefully removed. Reagent A was heated until melted. Then, 1000 μL of reagen<sup>t</sup> A was transferred into a 1.5 mL microtube and incubated for 10 min at 37 ◦C in a thermostatic water bath. Reagent B was added to the 1.5 mL microtube and mixed well. The solution was added to the slide, a coverslip was added, and the slide was incubated in the dark at 4 ◦C for 10 min until the gel solidified. The prepared sections were then incubated at 37 ◦C for 60 min in the dark. Fluorescence was visualized in 10 randomly selected fields of view for each cell slide at 40× magnification under a fluorescence microscope (Colibri 7, ZEISS, Jena, Germany). The fluorescence intensity of cells with active enzymes of MMP-2 or MMP-9 was quantified by the arithmetic mean intensity of ZEN 2.3 lite software. Each sample was assayed in triplicate, as described previously [29–31].

## *4.9. Rat Aortic Ring Assay*

Aortas were obtained from six-week-old Sprague–Dawley rats. Each aorta was cut into 1-mm slices and washed in sterile PBS three times, and then imbedded into 55 μL of Matrigel in 96-well plates. The aortic rings were then cultured in 100 μL of DMEM medium with 10% FBS and various concentrations of PSS and PSS with di fferent molecular markers. On day 7, the rings were photographed under a microscope (Colibri 7, ZEISS, Jena, Germany). The data obtained were analyzed and quantified using ImageJ software (ImageJ 1.8.0, Rawak Software Inc., Stuttgart, Germany). The animal experiments were approved by the Animal Ethics Committee of Marine Biomedical Research Institute of Qingdao (MBRI-2017-1106), and were strictly followed the guidelines of the institute.

### *4.10. Chick Chorioallantoic Membrane Assay*

Fertilized eggs were incubated in a constant-temperature incubator maintained at 37 ◦C and 40%–60% humidity for seven days. Gentle suction was applied to the hole located at the broad end of the egg to create a false air sac directly over the chick chorioallantoic membrane (CAM), and a 1–2 cm<sup>2</sup> segmen<sup>t</sup> was immediately removed from the eggshell. A round gelatin sponge (5 mm × 5 mm) saturated with PSS solution (100 or 200 μg/egg) or saline was placed into the area between the pre-existing vessels, and the embryos were further incubated for 48 h. The zones of neovascularization under and around the gelatin sponge were photographed under a stereomicroscope (SZX2-ILLT, OLYMPUS). The data obtained were analyzed and quantified using ImageJ software (ImageJ 1.8.0, Rawak Software Inc., Stuttgart, Germany).
