*4.3. Cell Culture*

The NIH/3T3 cell line was provided by the Core Facility of West China Hospital. The NIH/3T3 cell was cultured with DMEM (HyClone, Boston, MA, USA) supplemented with 10% fetal bovine serum (FBS) at 37 ◦C in a humidified atmosphere of 5% CO2.

#### *4.4. Cell Migration and Proliferation Assays*

Effects of Pa-THYs on NIH/3T3 cell migration were determined by wound healing assay. Briefly, wound healing assay was carried out in six-well plates (3 × 105 cells/well), wounds were created using a pipette tip. The cells were then rinsed with PBS to remove any free-floating cells and debris. After washing, the medium was replaced by a control medium with di fferent concentrations (0, 0.1, 1 and 10 μg/mL) Pa-THYs, and the cells were incubated at 37 ◦C. The area of wound healing was observed at 0, and 24 h, and representative images for each concentration were photographed. NIH/Image J software (https://imagej.net/NIH\_Image) was used for quantification of the scratch wound area based on an edge-detection and thresholding technique. We calculated the migrated area by calculating the blank area of the scratch in di fferent time point after Pa-THYs protein treatment.

E ffects of Pa-THYs on NIH/3T3 cell proliferation were determined by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (72 h) assays. Briefly, MTS assays were carried out in 96-well plates (2000 cells/well, five replicates), and the treatments of proteins were initiated at 24 h post-seeding, cells were cultured for 72 h. Then 20 μL MTS (Promega, Madison, WI, USA) was added to each well (100 μL medium) and incubated for 1–2 h at 37 ◦C. OD490 was measured by a Gen5 Microplate Reader (BioTek, Winooski, Vermont, USA) according to the manufacturer's instructions. IC50 values were calculated by GraphPad Prism 5 (San Diego, CA, USA).
