*4.3. Cell Culture and Treatments*

A dopaminergic cell line SH-SY5Y was used to establish an in vitro PD model. SH-SY5Ycells were kindly provided by Professor Ning Song (QingDao University) and maintained in Dulbecco's modified Eagle medium/F12 supplemented with 10% newborn calf serum (Gibco) in an incubator with an atmosphere of 5% CO2 at 37 ◦C. For all experiments, the cells were seeded on 96-well plates, 24-well plates or 6-well plates at a density of 1 × 10<sup>4</sup> cells–1 × 10<sup>5</sup> cells/mL for 24 h. Then the cells were incubated with MPP<sup>+</sup> for 30 min, then treated with different reagents for 24 h. The cells were divided into 12 groups. 1. NC group: treated with DMEM; 2: MPP group: treated with 100-μM MPP<sup>+</sup>; 3. UF1 group: treated with 100-μM MPP<sup>+</sup> and 100-μg/mL UF; 4. UF2 group: treated with 100-μM MPP<sup>+</sup> and 500-μg/mL UF; 5. UF3 group: treated with 100-μM MPP<sup>+</sup> and 800-μg/mL UF; 6. MA group: treated with 100-μM MPP<sup>+</sup> and 100-mM positive drugs Modopar; 7. NCLY group: treated with DMEM and 20-μM LY294002; 8: MPPLY group: treated with 100-μM MPP<sup>+</sup> and 20-μM LY294002; 9. UF1LY group: treated with 100-μM MPP<sup>+</sup>, 100-μg/mL UF and 20-μM LY294002; 10. UF2LY group: treated with 100-μM MPP<sup>+</sup>, 500-μg/mL UF and 20-μM LY294002; 11. UF3LY group: treated with 100-μM MPP<sup>+</sup>, 800-μg/mL UF and 20-μM LY294002; 12. MALY group: treated with 100-μM MPP<sup>+</sup>, 100-mM positive drugs Modopar and 20-μM LY294002. All the group had six wells and all the experiments were repeated three times in different batches of cells.

#### *4.4. Measurement of Cell Viability by MTT*

SH-SY5Y cells were plated at a density of 1 × 10<sup>4</sup> cells/100 μL in 96-well plates. Cell viability was quantitatively assessed using the MTT ([3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide]) assay [9]. After 24 h treatment, 20 μL MTT (0.5 mg/mL) regen<sup>t</sup> was added to each well and incubated at 37 ◦C for 4 h. The medium was removed and washed twice with phosphate buffer solution (pH 7.4), then 200 μL DMSO was added to solubilize the formazan crystals. Cell viability was measured at 494 nm by spectrophotometer (Bio-Tec Gen 5, Winooski, VT, USA). Unless stated otherwise, all other chemicals were purchased from Sigma-Aldrich.
