The 2,6-DCBQ standard was purchased from Aladdin (China). It was dissolved in methanol (HPLC grade, Fisher Scientific) to a stock concentration of 100 mM and stored in a sterile brown glass vial at −20˚C. The final methanol concentration in all treatment groups (including the control) was adjusted to 0.1% (v/v) by diluting the stock with culture medium. The control group received 0.1% methanol without 2,6-DCBQ to exclude solvent effects.

The human bladder epithelial cancer cell line T24 was obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China) and cultured in RPMI 1640 supplemented with 10% fetal bovine serum (Procell, Wuhan, China) and 1% penicillin/streptomycin (Invitrogen, USA), and placed in a cell incubator at 37˚C with 5% CO₂.

According to the manufacturer’s instructions, the cell viability was determined using the CCK8 cell proliferation-toxicity assay kit (DOJINDO, Japan). Specifically, T24 cells were seeded at a density of 7500 cells per well and cultured for 24 hours before cell viability testing. Based on previous research results [22], cells were treated with 10 - 250 μM 2,6-DCBQ for 24 hours, followed by the addition of CCK8 reagent and continued incubation in a 37˚C incubator for 1 hour. The absorbance was measured at 450 nm using a microplate reader. Based on the cell viability assay results, a curve depicting the changes in T24 cell viability under different concentrations of 2,6-DCBQ exposure was plotted, and the IC₅₀ was calculated. According to the IC₅₀ results, the subsequent exposure doses of 2,6-DCBQ were determined, with the high-dose group (H group) at IC₅₀, the intermediate-dose group (M group) at 1/2 IC₅₀, and the low-dose group (L group) at 1/4 IC₅₀.

T24 cells were seeded at a density of 7500 cells per well (100 μL), and after 24 hours of culture, the medium containing three different doses of 2,6-DCBQ was replaced and treated for another 24 hours. According to the manufacturer’s instructions, the cytotoxicity of 2,6-DCBQ was measured using a lactate dehydrogenase cytotoxicity assay kit (Beyotime, Shanghai, China).

The levels of malondialdehyde (MDA) adducts in T24 cells from three 2,6-DCBQ-treated groups were determined using a lipid oxidation (MDA) detection kit. T24 cells were seeded at a density of 7500 cells per well (100 μL) and cultured for 24 hours before the medium was replaced with one containing different doses of 2,6-DCBQ. After 24 hours of exposure to 2,6-DCBQ, cell samples were pretreated and MDA adducts were detected according to the instructions provided in the kit.

Transcriptome sequencing and data analysis were completed by Wuhan Kangce Technology Co., Ltd. (Wuhan, China).

2.5.1. RNA Extraction, Library Preparation, and Sequencing

Seed T24 cells in a 10 cm cell culture dish and replace the medium with one containing three different doses of 2,6-DCBQ after 24 hours of culture. After 24 hours of exposure to 2,6-DCBQ, total RNA was extracted from T24 cells using TRIzol Reagent (Invitrogen, USA) according to the method described by Chomczynski et al. [22]. After RNA extraction, DNaseI was used for DNA digestion. The integrity of RNA was determined by measuring the A260/A280 ratio using a NanodropTM OneC spectrophotometer (Thermo Fisher Scientific Inc, USA) and confirmed by 1.5% agarose gel electrophoresis. Finally, qualified RNA was quantified using the Qubit3.0 and QubitTM RNA Broad Range Assay kit (Life Technologies, USA).

According to the manufacturer’s instructions, the KCTM Stranded mRNA Library Prep Kit for Illumina® (Wuhan Kangce Technology Co., Ltd., China) was used to prepare a stranding RNA sequencing library using 2 μg of total RNA. The PCR products corresponding to 200 - 500 bps were enriched and quantified, and finally sequenced on a PE150 model DNBSEQ-T7 sequencing instrument (MGI Tech, China).

2.5.2. Transcriptome Data Analysis

1)Filteringandannotationofrawsequencingdata

The raw sequencing data were first filtered using Trimmomatic (version 0.36) to discard low-quality reads and trim reads contaminated by adapter sequences. Clean reads were then mapped to the reference genome of GRCh 38.87 using STRA software (version 2.5.3a). Reads mapped to the exon regions of each gene were counted using featureCounts (Subread-1.5.1; Bioconductor), and the number of reads per kilobase per million mapped sequences (RPKM) for each gene was calculated.

2)Differentiallyexpressedgenescreening

Use the edgeR package (version 3.12.1) in R language to screen genes with differential expression between groups, using raw count data as input. Genes with a false discovery rate (FDR) < 0.05 and |log₂ fold change| > 1 were considered significantly differentially expressed. The statistical significance of gene expression differences is determined by P < 0.05 and logFC > 1 or logFC < −1.

3)Pathwayanalysis

GO functional enrichment analysis and KEGG pathway analysis were conducted using KOBAS software (version: 2.1.1). GO enrichment annotations and KEGG pathway analysis results were obtained from biological process (BP), molecular function (MF), and cellular component (CC). Statistically significant enrichment was determined with an FDR < 0.05 (replacing the original P-value-only threshold).

To test the effect of 2,6-DCBQ on the viability of T24 cells and to explore suitable exposure concentrations for subsequent experiments, we assessed the changes in T24 cell viability after exposure to 10 - 250 μM 2,6-DCBQ for 24 hours, based on previous research findings. The results are shown in Figure 1(a). Exposure to 2,6-DCBQ significantly reduced the cell viability of T24 cells, and this reduction was dose-dependent. The IC50 calculated from the T24 cell viability curve after 24 hours of exposure to 2,6-DCBQ was 114.0 (109.6 - 118.0) μM. To set approximate low, medium, and high exposure doses, we selected 25 μM, 50 μM, and 100 μM, which roughly correspond to 1/4 IC₅₀ (28.5 μM), 1/2 IC₅₀ (57 μM), and IC₅₀ (114 μM), respectively, given the convenience of dose preparation and consistency with previous studies. These doses are referred to as low-dose (L, 25 μM), medium-dose (M, 50 μM), and high-dose (H, 100 μM) groups throughout the study, and subsequently measured lactate dehydrogenase (LDH) activity. The results are shown inFigure 1(b). As the concentration of 2,6-DCBQ increased, the activity of LDH gradually increased (P < 0.01), indicating that within the exposure range of 0 - 100 μM, 2,6-DCBQ caused T24 cell death in a dose-dependent manner. In summary, 2,6-DCBQ has a significant toxic effect on T24 cells, and this effect is dose-dependent.

Figure 1. Reduced viability and oxidative damage in T24 cells after 24 hours of exposure to 2,6-DCBQ. Note: (a) represents the viability curve of T24 cells after exposure to 2,6-DCBQ at different concentrations. (b) illustrates the percentage of cytotoxicity calculated based on LDH enzyme activity after exposure to 2,6-DCBQ, where the percentage of cytotoxicity (%) = (absorbance of treated sample − absorbance of sample control well)/(absorbance of maximum enzyme activity of cells − absorbance of sample control well) × 100. (c) shows the changes in MDA adduct levels in each exposure group relative to the control group after exposure to 2,6-DCBQ. All values are expressed as mean ± standard deviation. Each experiment was set up with six biological replicates, and one-way ANOVA was used for inter-group difference analysis, followed by Dunnett’s test for pairwise comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

We further examined the content of malondialdehyde (MDA) adducts in T24 cells after exposure to 2,6-DCBQ. The results are shown in Figure 1(c). Compared with the control group (1.00 ± 0.05), the MDA adduct levels in the H group (1.81 ± 0.34) were significantly increased (P < 0.05), while there was no statistical difference in MDA adduct levels between the L and M groups. This result suggests that at high doses, 2,6-DCBQ causes lipid peroxidation damage in T24 cells, but such direct oxidative damage was not detected at lower doses under the present assay conditions.

Transcriptome sequencing was performed on T24 cells exposed to different doses of 2,6-DCBQ for 24 hours. The quality of the sequencing data is shown in Table 1. The clean reads obtained from each sample ranged from 69,082,156 to 199,819,894, and the proportion of bases with quality greater than Q30 in the clean reads was greater than 97.8%.

Table 1.Summary of transcriptome sequencing data.

Under the principal component scores of 21.57% and 16.11%, it can be observed that the control group and the three 2,6-DCBQ exposure groups are clearly separated, suggesting that 2,6-DCBQ exposure causes changes in the transcriptome of T24 cells. Cluster analysis was performed on the differentially expressed genes of all samples. Firstly, the samples from each group were well clustered together. Additionally, we found that the direction of differential gene expression changes in Group L and Group M was similar, while it differed from the gene expression pattern in the highest dose Group H. Volcanoes plots were used to show the changes in gene expression under different doses of 2,6-DCBQ exposure. Compared to the control group, 963, 1180, and 979 genes were significantly upregulated, and 366, 409, and 795 genes were significantly downregulated (FDR < 0.05, |log₂FC| > 1) after exposure to low, intermediate, and high doses of 2,6-DCBQ, respectively. In summary, we obtained high-quality gene expression data of T24 cells through transcriptome sequencing, and found that 2,6-DCBQ exposure leads to significant changes in the transcriptome of T24 cells.

To further investigate the alterations in biological functions of T24 cells after exposure to DCBQ, we conducted GO functional classification annotation and KEGG pathway analysis on the differentially expressed genes of T24 cells after exposure to 2,6-DCBQ. In the GO functional classification annotation, the upregulated differentially expressed genes in Group L, M, and H were enriched in 304, 261, and 310 gene-related functions, respectively, while the downregulated differentially expressed genes were enriched in 273, 281, and 339 gene-related functions, respectively. Venn diagrams were used to statistically analyze the common gene-related functions enriched by the upregulated and downregulated differentially expressed genes in the three exposure groups. Among them, there were 42 common gene-related functions enriched by the upregulated differentially expressed genes in the three exposure groups, and 49 common gene-related functions enriched by the downregulated differentially expressed genes. Among the 42 common gene-related functions enriched by the upregulated differentially expressed genes, we identified multiple functions related to mitochondrial autophagy, such as mitochondrial autophagy, positive regulation of autophagy, autophagosome, autophagosome assembly, macroautophagy, and autophagy. Among the 49 common gene-related functions enriched by the downregulated differentially expressed genes, we identified multiple functions related to insulin, such as insulin-like growth factor I binding, insulin-like growth factor II binding, regulation of the insulin-like growth factor receptor signaling pathway, negative regulation of the insulin-like growth factor receptor signaling pathway, positive regulation of the insulin-like growth factor receptor signaling pathway, and proliferation of type B pancreatic cells.

Similarly, in the KEGG pathway analysis, the upregulated differential genes in Group L, Group M, and Group H enriched 24, 25, and 31 gene-related pathways, respectively, while the downregulated differential genes enriched 24, 25, and 21 gene-related pathways, respectively. Venn diagrams were used to statistically analyze the common gene-related pathways enriched by the upregulated and downregulated differential genes in the three exposure groups. Among them, the three exposure groups had 6 common gene-related pathways enriched by the upregulated differential genes and 3 common gene-related pathways enriched by the downregulated differential genes. The common gene-related pathways enriched by the upregulated differential genes included mitochondrial autophagy-animal, ferroptosis, autophagy-animal, ABC transporter, ECM-receptor interaction, complement, and coagulation cascade, while the common gene-related pathways enriched by the downregulated differential genes included mitochondrial autophagy-animal, autophagy-animal, and pathogenic Escherichia coli infection. In summary, through GO functional enrichment analysis and KEGG pathway analysis, we found that after exposure to 2,6-DCBQ, the expression of mitochondrial autophagy-related genes in T24 cells was upregulated, while the expression of insulin-related genes was downregulated (Figure 2).

Figure 2. GO functional enrichment analysis of each dose group. Note: (a) represents the Venn diagram for the statistical analysis of the number of KEGG pathways associated with upregulated differential genes across various dose groups. (b) represents the Venn diagram for the statistical analysis of the number of KEGG pathways associated with downregulated differential genes across various dose groups. (c) represents the KEGG pathway analysis diagram for upregulated differential genes across various dose groups. (d) represents the KEGG pathway analysis diagram for downregulated differential genes across various dose groups. L: Low-dose 2,6-DCBQ exposure group; M: Medium-dose 2,6-DCBQ exposure group; H: High-dose 2,6-DCBQ exposure group.