## 3 Results
### PSEFS Suppresses the Phosphorylation of p38 MAPK in T24 Cells  
Several studies demonstrated that cytoplasmic early signaling molecules, such as MAPKs (p38 MAPK, JNK, and ERK) and AKT, play crucial roles in mediating the progression and development of bladder tumors [6; 7; 8]. Therefore, we investigated whether the phosphorylation levels of AKT and MAPKs are affected by PSEFS in T24 cells. PSEFS treatment induced phosphorylation of p38 MAPK in T24 cells, whereas levels of phosphorylated ERK, JNK, and AKT remained unchanged (Figure 3A-D). To further analyze the direct action of PSEFS on p38 MAPK phosphorylation, we employed the p38 MAPK inhibitor SB203580, finding that SB203580 treatment effectively attenuated phosphorylation of p38 MAPK in PSEFS-treated T24 cells (Figure 3E). In addition, SB203580 treatment reversed PSEFS effect on T24 cell proliferation (Figure S3A,B). These results suggested that PSEFS might stimulate the growth inhibition of T24 cells by inducing p38 MAPK phosphorylation. Figure 2: Involvement of cell cycle regulators in PSEFS-mediated cell cycle arrest at the G1 phase. (**A**,**B**) Immunoblot images of CDK4, cyclin D1, CDK2, cyclin E, p21 WAF1, p53, and p27 KIP1 after treatment of T24 cells with PSEFS for 24 h. GAPDH was used as an internal control. (**C**) Cell lysates were immunoprecipitated with anti-CDK4 and anti-CDK2 antibodies, followed by immunoblot with the anti-p21 WAF1 antibody. Data represent the mean \(\pm\) SD of triplicate experiments. \({}^{*}p<0.05\), \({}^{**}p<0.01\) vs. control. The MMP-9-Related Transcription Factors SP1, AP-1, and NF-\(\kappa\)B are Associated with PSEFS-Induced Mitigation of T24 Cell Migration and Invasion  
To determine the migration and invasion capability of PSEFS-treated T24 cells, we performed both wound-healing and invasion assays. We evaluated migration potential using a wound-healing assay by shifting the cells into the wound region. Wound closure by T24 cells was hindered by addition of PSEFS (Figure 4A). Additionally, we estimated the invasive ability of T24 cells according to their potential to invade transwell plates using a Boyden chamber assay. Compared with untreated cells, we found that PSEFS treatment prevented cells from crossing the transwell basement membrane (Figure 4B). A recent study reported that the activity of MMPs, including MMP-9 and MMP-2, decomposes the extracellular matrix, thereby promoting the invasiveness and migratory ability of bladder cancer cells [13; 14; 15]. Therefore, we performed a gelatin zymography assay to clarify the enzymatic activities of MMP-9 and MMP-2 in PSEFS-treated T24 cells. As shown in Figure 4C, PSEFS treatment blocked MMP-9 activity in T24 cells, although MMP-2 activity remained unchanged, and we observed attenuated levels of MMP-9 protein. Many studies have shown that MMPs were regulated by heat shock proteins [30; 31; 32]. Therefore, we investigated whether PSEFS suppresses the expression level of HSP70 in T24 cells. Immunoblot analysis revealed that PSEFS treatment did not affect the HSP70 level in T24 cells (Figure S4B). Finally, to determine the regulatory mechanism of PSEFS-mediated inhibition of MMP-9 activity/expression, we performed an electrophoretic mobility shift assay (EMSA) using oligonucleotides comprising common sequences targeted by SP1, AP-1, and NF-\(\kappa\)B. The EMSA results indicated that PSEFS disrupted the binding of each transcription factor to the cis-element of MMP-9 in T24 cells (Figure 4D). These data indicated that PSEFS might impede the migratory potential and invasive ability of T24 cells in part by reducing MMP-9 activity mediated by transcription factors. Figure 3: PSEFS promotes the phosphorylation of p38 MAPK in T24 cells. (**A–D**) Changes in phosphorylated levels of MAPKs (JNK, ERK1/2, and p38 MAPK) and AKT according to immunoblot. (**E**) Verification of p38 MAPK phosphorylation using SB203580 (a specific p38 MAPK inhibitor). Bar graphs show fold-changes in expression relative to controls. Data represent the mean \(\pm\) SD of triplicate experiments.