*2.8. Scanning and Transmission Electron Microscopy*

The human bladder cancer J82 cells were inoculated at a density of 2 × 104 cells/mL on slides attached to a 24-well plate, and then treated with the drug at a concentration of 10 μM. After being cultured in the incubator for 24 h, the cells were taken out and washed with precooled PBS twice. The scabertopin-treated cells were fixed with 4% paraformaldehyde (Beyotime Biotechnology, P0099-500 mL) at room temperature and dehydrated with alcohol. Then, their morphology was observed using a scanning electron microscope (SEM) (Regulus8100, HITACHI, Tokyo, Japan).

The J82 cells were inoculated into 6-well plates and then treated with gradient concentrations of scabertopin for 24 h. The culture medium was discarded. The cells were treated with an electron microscope fixative (Servicebio Technology, Wuhan, China, G1102-100 mL) for 2–4 h at 4 ◦C, collected, and then centrifuged at low speed until green bean-sized clumps of cells could be seen at the bottom of the tubes. After further wrapping, postfixation, dehydration, permeabilization, embedding, sectioning, and staining, the cells were observed under a transmission electron microscope (TEM) and images were collected for analysis.

#### *2.9. Measurement of Reactive Oxygen Species*

Intracellular ROS levels were measured using an intracellular ROS assay kit (Solarbio Science & Technology, Beijing, China, CA1420). The J82 cells were treated with gradient concentrations of scabertopin for 24–48 h. In the rescue experiments, the cells were incubated with scabertopin for 2 h with 5 mM of ROS scavenger *N*-acetylcysteine (NAC) (MedChemExpress, Monmouth Junction, NJ, USA, HY-B0215). The cells were washed three times with PBS and treated with 10 μM of dichlorofluorescein diacetate probe for 20–30 min. Subsequently, the cells were washed three times with a serum-free medium, and their fluorescence intensity was measured using a fluorescence microplate reader (BioTek, Winooski, VT, USA, Synergy H1).

In order to determine the type of ROS, the J82 cells were treated with gradient concentrations of scabertopin and then loaded with dihydroethidium (DHE) probe (KeyGEN BioTECH, KGAF019) at a concentration of 25 μM and incubated at 37 ◦C for 60 min in the darkness. After the incubation, the cells were washed with a fresh culture medium and their fluorescence intensity was measured using a fluorescence microplate reader (BioTek, Winooski, VT, USA, Synergy H1). The cells were then imaged under an inverted fluorescence microscope.

#### *2.10. Mitochondrial Membrane Potential Assay*

The effect of scabertopin on mitochondrial membrane potential (ΔΨ) was detected using the Mitochondrial membrane potential assay kit JC-1 (Beyotime Biotechnology, C2006). The cells were treated with gradient concentrations of scabertopin, washed with PBS, added to a cell culture medium, mixed with the JC-1 solution, and incubated at 37 ◦C for 20 min according to the manufacturer's instructions. The fluorescence intensity of the cells was measured using a fluorescence microplate reader (BioTek, Winooski, VT, USA, Synergy H1), and the cells were imaged under an inverted fluorescence microscope. Whereas green fluorescence is an indicator of depolarized mitochondria, intact mitochondria produce red fluorescence.

When ΔΨ is at higher levels, JC-1 aggregates in the matrix of mitochondria to form polymers and produce red fluorescence. When ΔΨ is at a lower level, JC-1 cannot aggregate in the mitochondrial matrix. At this time, JC-1 is monomer and can produce green fluorescence. The ratio of monomer/polymer represents the ratio of green fluorescence/red fluorescence, which can be used to measure the proportion of mitochondrial depolarization

#### *2.11. GSH Assay*

The GSH (reduced glutathione) and GSSG (oxidized glutathione disulfide) assay kit (Beyotime Biotechnology, S0053) is a simple and easy-to-use assay that can detect the contents of GSH and GSSG, respectively. Briefly, total GSH and GSSG levels were measured at a wavelength of 412 nm after the cells were treated with gradient concentrations of scabertopin. The level of reduced GSH was calculated according to the following formula:

$$GSH = \text{total glutathiline} \left( GSH + GSSG \right) - 2 \times GSSG \tag{2}$$

#### *2.12. Wound Healing Assay*

A total of 70 <sup>μ</sup>L of J82 cells were inoculated at a density of 8 × <sup>10</sup><sup>5</sup> cells/mL into each insert of a Culture-Insert 2 Well (ibidi, Grafelfing, Germany, 80206) in the middle of a dish. After the cells were attached, the insert was removed with forceps and the old medium was aspirated off. The cells were washed gently 1–2 times with PBS, treated with different concentrations of the drugs, placed in the incubator for further incubation, and removed at 0 and 24 h for fluorescent inverted microscopy (IX71, Olympus, Tokyo, Japan) to observe whether the peripheral cells had migrated to the central scratch area. The cells were photographed and recorded. The percentage of wound healing was analyzed using ImageJ software and calculated as the ratio of the initial scratch area minus the partially healed area that had healed at a certain time to the initial area, according to the following formula.

$$\text{initial area} - \frac{\text{area at a certain time point}}{\text{initial area}} \tag{3}$$

#### *2.13. Transwell Assay*

The J82 cells treated with different concentrations of drugs for 24 h were collected and their density was adjusted to 5 × <sup>10</sup><sup>4</sup> cells/well. The cells were inoculated into the upper chamber of a transwell plate and the lower chamber was supplemented with 10% FBS medium. The plate was placed in the incubator for 24 h, washed twice with PBS, and fixed with methanol. Then, the cells were treated with 0.1% Giemsa staining solution, washed three times with PBS, and allowed to air dry. The number of migrated cells was recorded by photography under multiple high-magnification fields using a microscope (Etaluma, Inc., San Diego, CA, USA, LS720) and by counting the number of migrated cells.

#### *2.14. Cell cycle Assays*

The J82 cells were treated with different concentrations of drugs for 24 h, digested with tryspin, collected, and washed 1–2 times with PBS. The cells were fixed by adding precooled 70% ethanol and then washed with PBS to remove the fixative. The cells were administered with a RNase/propidium iodide (PI) staining working solution and incubated for 30 min at room temperature under protection from light. The samples were subjected to flow cytometry (FACSCanto, Becton, Dickinson and Company, Franklin Lakes, NJ, USA), and Modfit software was used to analyze the results.

#### *2.15. Cell Apoptosis Analysis*

The J82 cells were treated with different concentrations of drugs for 24 h, digested by using EDTA-free trypsin, collected, washed and resuspended with PBS, and centrifuged to collect cell precipitates. The precipitates were resuspended again with a small amount of a binding buffer, mixed with an Annexin-V-FITC working solution, incubated for 5 min at room temperature under protection from light, and mixed with a PI reagent and PBS. The samples were analyzed using flow cytometry and the results were analyzed using Modfit software.

#### *2.16. Western Blot Analysis*

A total of 10 μL of ProteinSafe™ Phosphatase inhibitor cocktail (DI201, TransGen Biotech, Beijing, China) and ProteinSafe™ Protease inhibitor cocktail (DI101, TransGen Biotech, Beijing, China) was added to every 1 ml of ProteinExt® Mammalian total protein extraction kit (TPEB) (DE101, TransGen Biotech, Beijing, China). The TPEB mixed reagents were made by mixing the above three reagents according to the protocols. The cells were mixed with the TPEB mixed reagents on ice after 24 h of scabertopin treatment. The lysates were collected and centrifuged at 14,000× *g* for 10 min at 4 ◦C. Protein concentrations were analyzed using a Bicinchoninic acid (BCA) kit (P0010, Beyotime Biotechnology). Equal amounts of protein samples were separated through electrophoresis on a 10% precast gel (M00664, GenScript Biotech, Nanjing, China). The proteins were then transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk at 4 ◦C overnight. Afterward, the membranes were incubated for 2 h at room temperature with different primary antibodies, including Bax (1/10,000), Bcl-2 (1/10,000), caspase-9 (1/1000), caspase-3 (1/1000), caspase-8 (1/1000), GAPDH (1/10,000), MMP-9 (1/500), PI3K (1/1000), p-PI3K (1/1000), AKT (1/1000), p-AKT (1/1000), glutathione peroxidase 4 (GPX4, 1/1000), gasdermin-D (GSDMD, 1/1000), RIP (1/1000), p-RIP (1/1000), MLKL (1/1000), p-MLKL (1/1000), RIP3 (1/1000), and p-RIP3 (1/1000). The membranes were washed three times with TBST and incubated with the secondary antibodies (1/2000–1/20,000) for 1 h at room temperature. The target protein lane was imaged using an iBright 1500 (Invitrogen) with enhanced chemiluminescent substrates (Merck Millipore, Darmstadt, Germany, WBKLS0500). Original blots see Supplementary File S1.

### *2.17. Statistical Analysis*

Statistics and bar graphs were analyzed using the Xiantao Academic Online Tools (https://www.xiantao.love/products) (accessed on 30 August 2022), which is based on R and the ggplot2 R package. The means of two groups were considered significantly different if \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001.

#### **3. Results**

#### *3.1. Bladder Cancer Cells Are Sensitive to Scabertopin*

The chemical structure of scabertopin is shown in Figure 1A. The two boxes show the α-methylene-γ-lactone and butenolide moieties that confer the drug activity. The FTIR spectrum of scabertopin is shown in Figure 1B, and the UV absorption peaks of scabertopin in the DMEM medium at 0, 24, and 48 h are shown in Figure 1C. According to the Beer– Lambert law, when the length of the absorption cell, the light source, and the type of the substance to be measured are the same, the absorbance is strictly proportional to the concentration of the substance. Since the intensity of the UV maximum absorption peaks of scabertopin at 0, 24, and 48 h were all around 0.55, the concentration of scabertopin did not change. In conclusion, it can be considered that scabertopin can maintain stability in a DMEM medium. The viability of scabertopin-treated bladder cancer cells (T24, J82, RT4, and 5637) and human ureteral epithelial immortalized cells (SV-HUC-1) was determined using the CCK-8 assay. The results showed that scabertopin significantly inhibited the viability of the human bladder cancer cells (J82, T24, RT4, and 5637) in a dose-dependent manner (Figure 1D). The 24 h IC50 of scabertopin for the bladder cancer cell lines was approximately 20 μM, and the 48 h IC50 was even lower (approximately 18 μM). However, the IC50 values of scabertopin for the SV-HUC-1 cells at 24 and 48 h were 59.42 and 55.84 μM, which were considerably higher than those for the bladder cancer cells (Figure 1D). Next, the J82 cell line was arbitrarily selected for further study. In the following section, if the treatment time of scabertopin is not explicitly mentioned, it defaults to 24 h.

**Figure 1.** Chemical structure, stability, and efficacy of scabertopin against bladder cancer cells. (**A**). Chemical structure scabertopin. The two boxes show the structure of the molecule with the drug activity of α-methylene-γ-lactone and butenolide. (**B**). Infrared spectrum of scabertopin. The stretching vibration of =CH2 exists at 3080 cm<sup>−</sup>1, and the carbonyl stretching vibration of α-methyleneγ-lactone structure exists at 1760 cm−1. The peak at 1710 cm−<sup>1</sup> is the carbonyl stretching vibration of another non-lactone α,β-unsaturated ester, and the C=C stretching vibration exists at 1650 cm<sup>−</sup>1. (**C**). The UV absorption peak of scabertopin in the DMEM medium at 0, 24, and 48 h. (**D**). 24 and 48 h IC50 of bladder cancer cell lines (J82, T24, 5637, and RT4) and human ureteral epithelial immortalized cells (SV-HUC-1) (n = 4).
