*2.2. Material Preparation*

Scabertopin (10 mg) was dissolved in 1 ml of dimethyl sulfoxide DMSO (Beyotime Biotechnology, ST2335-100 mL) and configured into a master batch with a concentration of 40 μM such that the maximum concentration of DMSO in the cell culture medium was 0.143%. In the subsequent experiment, in order to exclude the effect of DMSO on cells, 0.143% DMSO was contained in all negative control group media as control. A total of 10 μL of scabertopin and deoxyelephantopin solution was applied evenly on KBr-pressed plates, evaporated and dehydrated using an infrared lamp, and analyzed using a Fouriertransform infrared (FTIR) spectroscopy and KBr-pressed plates. KBr (P116274-100 g) was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China).

#### *2.3. Infrared Spectroscopy*

A Fourier-transform infrared tester (Nexus-670 produced by Nexus, Madison, WI, USA) was used to conduct IR spectrum test on the scabertopin powder: the wavelength

range was 400–4000 cm<sup>−</sup>1, the number of scans was 32, and the resolution was 4 cm−1. The operation was as follows: grind KBr dried under vacuum for 8 h at 140 ◦C, take 10 mg and press into tablets to obtain background tablets, and grind another 10 mg of KBr and 1 mg of scabertopin powder in an agate mortar evenly. The IR spectroscopy was performed on the obtained background pieces (only KBr) and the samples (KBr and scabertopin powder).

#### *2.4. UV Absorption Assay*

The mixed solution of scabertopin and DMEM medium was tested using an UV-Vis spectrophotometer (UV9100B from Beijing LabTech Instrument Co., Ltd., Beijing, China), with a wavelength range of 200–360 nm and a resolution of 2 nm.

If the chemical structure of the substance were stable, its UV maximum absorption wavelength would not change. It could be seen from the UV spectrum that the UV maximum absorption peak of scabertopin is around 214 nm, which is consistent with the UV spectrum of the α,β-unsaturated carbonyl moiety. There was no change at 0, 24, and 48 h, and it could be inferred from the Beer–Lambert law that, when the length of the absorption cell, the light source, and the type of the substance to be tested are the same, the absorbance is strictly proportional to the substance concentration.

$$Abs = \lg\left(\frac{1}{T}\right) = Kcl\tag{1}$$

*Abs*: Absorbance; *<sup>T</sup>*: transmittance; *<sup>K</sup>*: molar extinction coefficient, cm2·mol; *<sup>c</sup>*: molar concentration, mol/L; and *l*: the length of the absorption cell, cm.

#### *2.5. Cell Lines and Culture*

Human bladder cancer cell lines (J82 and T24) and human ureteral epithelial immortalized cells (SV-HUC-1) were purchased from the ATCC cell bank, and 5637 and RT4 cell lines were purchased from the China Centre for Type Culture Collection (Shanghai, China). All cell culture media and FBS (30044333) were obtained from Gibco. J82 cells were cultured in DMEM (10% FBS) medium, T24 and 5637 cells were cultured in RPMI 1640 (10% FBS), RT4 cells were cultured in McCoy's 5A (Gibco, 16600082) (10% FBS) medium, and SV-HUC-1 cells were cultured in F-12K (Gibco, 21127022) (10% FBS) medium. All cell lines were grown in a cell culture incubator at 37 ◦C and 5% CO2 to maintain cell growth. The cells were revived and passaged two times for subsequent experiments.

#### *2.6. CCK-8 Assay*

The human bladder cancer cells (J82 and T24) and human ureteral epithelial immortalized cells (SV-HUC-1) were inoculated into 96-well plates (100 μL per well). Subsequently, after being added with different concentrations of drugs, the plates were incubated in an incubator at 37 ◦C and 5% CO2 for 24–48 h. A total of 10 μL of CCK-8 reagent (Dojindo Laboratories, CK04) was added to each well, and incubation was continued in the incubator for 1–4 h. Finally, absorbance was detected at 450 nm by using an enzyme marker (Bio-Rad, Hercules, CA, USA). The experimental data (mean ± standard deviation) were plotted in the form of histograms. Dose–response curves were generated using GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA) software, and the absolute 50% inhibitory concentration (IC50) was determined.

#### *2.7. LIVE/DEAD Cell Activity Assay*

The J82 cells were inoculated at a density of 1 × 104 cells/mL into 96-well plates and treated with gradient concentrations of scabertopin for 24–48 h. Subsequently, the cells were washed with phosphate buffered saline (PBS) in accordance with the instructions of the LIVE/DEAD cell vitality assay kit (Invitrogen), and each group was incubated for 30 min at room temperature with 5 μL of the supplied 4 mM calcein AM stock solution to the 10 mL ethidium dimers (EthD-1). The cells were observed and photographed with an inverted fluorescence microscope, which showed live cells in green (AM) and dead cells in red (EthD-1).
