## 3 Results  
PSEFS Prohibits the Proliferation of Bladder Cancer T24 Cells by Stimulating Cell Cycle Arrest at the G1 Phase  
To investigate the inhibitory effect of PSEFS on the proliferation of bladder cancer T 24 cells, we employed both cell-counting and MTT assays. Trypan Blue staining showed that treatment of T24 cells with PSEFS revealed concentration- and time-dependent decrease in the number of viable cells compared to that of the control (Figure 1B and Figure S1B), with the results of MTT assays supporting these results (Figure 1A and Figure S1A). In addition, up to 800 ug/mL, PSEFS treatment had no effect on growth inhibition of BdFC normal bladder cells (Figure S2A,B). In proliferation assays, we determined an IC\({}_{50}\) of 800 ug/mL in T24 cells and observed both decreased cell number and diminished cell density according to phase-contrast microscopy (Figure 1C). Moreover, generation of fluorescence-activated cell sorting (FACS) histograms to determine whether PSEFS-induced anti-proliferative effects in T24 cells were related to changes in cell cycle progression indicated that PSEFS treatment induced G1-phase arrest (Figure 1D-H). To investigate the cell death level in PSEFS-treated T24 cells, ELISA-basedapoptosis assay was performed. PSEFS treatment did not affect cell death in T24 cells (Figure S4A). These results indicated that PSEFS treatment enhanced the proliferative inhibition of bladder cancer T24 cells by inducing cell cycle arrest at the G1 phase.  
PSEFS Stimulates G1-Phase Arrest of the Cell Cycle Via Reduced Expression of CDKs and Cyclins by Increasing p21\({}^{\text{WAF1}}\) Protein Level  
To determine the precise mechanism associated with cell cycle arrest at the G1 phase in PSEFS-treated T24 cells, we evaluated the levels of cell cycle-related proteins positively modulating the transition from G1 to S phase. The results showed that PSEFS treatment reduced the expression level of CDK4 and its counterpart cyclin D1 in T24 cells (Figure 2A). Additionally, levels of both CDK2 and cyclin E decreased in the presence of PSEFS (Figure 2A). We then examined levels of p21\({}^{\text{WAF1}}\) and p27\({}^{\text{KIP1}}\) as negative regulators of the cell cycle in PSEFS-treated cells. We found that PSEFS treatment upregulated p21\({}^{\text{WAF1}}\) levels with no effect on p27\({}^{\text{KIP1}}\) levels or levels of the tumor suppressor p53 (Figure 2B). Because p21\({}^{\text{WAF1}}\) is reportedly a critical regulator of cell cycle transition from G1 to S phase, we performed immunoprecipitation (IP) experiments using the anti-CDK2 and anti-CDK4  
Figure 1: Peanut sprout extracts cultivated with fermented sawdust medium (PSEFS) inhibits T24 cell proliferation via G1-phase arrest. (**A**) Cells were exposed to different concentrations of PSEFS (0, 400, 600, and 800 ug/mL) for 24 h, and an MTT assay was performed to evaluate cell viability. (**B**) Cell counting was performed to determine cell growth. (**C**) Morphological changes of cells according to phase-contrast microscopy (40\(\times\)). (**D–G**) The cell cycle distribution of T24 cells treated with the indicated concentrations of PSEFS for 24 h and evaluated by flow cytometry analysis. (**H**) The percentage of the cell population in specific cell cycle phases. Data represent the mean \(\pm\) SD of triplicate experiments. \({}^{*}\)\(p<0.05\), \({}^{**}\)\(p<0.01\) vs. control.  
antibodies and detected immunocomplex formation by immunoblot using an anti-p21 WAF1 antibody. IP experiments indicated that PSEFS treatment markedly upregulated p21 WAF1 levels according to the observed complex formation with both CDK4 and CDK2 in T24 cells (Figure 2C). These results suggested that PSEFS elicited G1-phase cell cycle arrest through suppression of cyclin and CDK expression via upregulated p21 WAF1 levels.