*3.6. Cytotoxicity Assay*

HepG2 (liver cancer cell line) and Hela (cervical cancer cell line) were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. The cells were grown in an RPMI-1640 culture medium. Cytotoxicity against HepG2 cells and HeLa cells was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, Missouri, St. Louis, MO, USA) method, as described previously [45]. In addition, 5-fluorouracil (5-FU) (Beijing Solarbio Science and Technology Co., Ltd., 99.8%) (Beijing, China) and adriamycin (Shanghai Macklin Biochemical Co., Ltd, 99.8%) (Shanghai, China) were used as positive controls, respectively.

#### *3.7. Isolation and Culture of Spleen Lymphocytes*

The BALB/c female mice were sacrificed via cervical dislocation, and their spleens were removed aseptically. The splenocytes were washed using RPMI1640 supplemented with penicillin/streptomycin (100 U/mL and 100 μg/mL, respectively) and 10% heatinactivated FBS, and collected in a centrifuge tube. The erythrocytes were removed for 3 min with an erythrocyte lysis buffer. The cells were plated at a density of 5 × 10<sup>6</sup> cells/mL or 1 × 10<sup>7</sup> cells/mL. Cell numbers were performed using a hemocytometer, and cell viability was determined using the trypan-blue dye exclusion technique; cell viability showed more than 95%. The culture media were kept in a humidified atmosphere of 5% CO2 at 37 ◦C.

#### *3.8. Cell Activity and Cell Proliferation*

In each 96-well cell culture plate, 100 μL of lymphocyte suspension was inoculated with a concentration of 1\*10<sup>7</sup> cells/mL in each well, and the culture was left overnight in a 37 ◦C, 5% CO2 incubator to stabilize the cells. Then, the compounds or positive control (CsA) diluted in a complete medium to different concentrations were added to each well, resulting in the final concentrations of 1, 5, 10, 15, 20, 30, and 40 μM, respectively. The final concentrations of the compounds in the anti-proliferation assay were 20 μM, 35 μM, 50 μM, 70 μM, and 100 μM. After 72 or 48 h incubation in the incubator, the effect of the compounds on the survival rate and anti-proliferation of splenocytes was analyzed using the CCK-8 method.
