Fig 2: Effect of PAGE4 knockdown on cell proliferation and oxidative stress level of WPMY-1 and PrPF cells under OS. (a) qRT-PCR was used to verify the knockdown efficiency of different siRNAs on PAGE4 in WPMY-1 and PrPF cells at the transcriptional level. (b) The cell viability of WPMY-1 and PrPF after knockdown of PAGE4 with or without 400 µM H2O2 treatment at different time points by CCK-8 assay. qRT-PCR was used to verify the effect of combined or noncombined 400 µM H2O2 on the transcription level of (c) SOD1 and (d) CAT after knockdown of PAGE4 in WPMY-1 and PrPF cells. (e) Immunoblot assay shows the effects of PAGE4 knockdown with or without 400 µM H2O2 on the protein expression levels of SOD1 and CAT in WPMY-1 and PrPF cells. (f) DCFH-DA fluorescent probe was used to analyze the accumulation of ROS by PAGE4 knockdown in WPMY-1 and PrPF cells under OS, and different color curves were used to represent different treatment groups. (g) Statistical analysis of mean fluorescence intensity (MFI) of DCFH-DA in WPMY-1 and PrPF cells after different treatments. GAPDH is used as loading control. ns: no significant difference. In (b), (c), (d), and (g), *: si-con vs. si-con + H2O2; red asterisk: si-con + H2O2 vs. si-PAGE4-1 + H2O2; blue asterisk: si-con + H2O2 vs. si-PAGE4-2 + H2O2; *: p < 0.05; **: p < 0.01; ***: p < 0.001. ANOVA or Student's t-test.

Fig 3: The effects of knockdown of PAGE4 on MAPK signaling pathways of WPMY-1 and PrPF cells under OS. (a) Immunoblot assays showing the effects of PAGE4 knockdown with or without 400 µM H2O2 on the protein expression levels of p-ERK1/2, t-ERK1/2, p-JNK1/2, and t-JNK1/2 in WPMY-1 and PrPF cells. (b) Relative protein expression abundance of p-ERK/t-ERK in different treatment groups. (c) Relative protein expression abundance of p-JNK/t-JNK in different treatment groups. GAPDH is used as loading control. ns: no significant difference; *: si-con vs. si-con + H2O2; red asterisk: si-con + H2O2 vs. si-PAGE4-1 + H2O2; blue asterisk: si-con + H2O2 vs. si-PAGE4-2 + H2O2; *: p < 0.05; **: p < 0.01; ***: p < 0.001. ANOVA or Student's t-test.

Fig 4: The expression of PAGE4 in prostate. (a) qRT-PCR analysis demonstrating the expression level of PAGE4 gene in BPH tissue (n = 10) and normal prostate tissue (n = 10) (b) Immunoblot assay revealed the protein expression of PAGE4 in normal prostate tissues and BPH tissues. Immunolocalization of PAGE4 for (c) normal prostate tissues and (d) BPH tissues. DAPI (blue) shows nuclear staining; Cy3-immunofluorescence (red) shows PAGE4 protein; a-SMA (green) indicates stromal staining. Sections of all samples were used for immunofluorescence experiments, and representative graphs were selected for figures. GAPDH is used as loading control. ***: p < 0.001. Student's t-test. The scale bars are 100 µm.

Fig 5: The effects of overexpression of PAGE4 on apoptosis and cell cycle of WPMY-1 and PrPF cells under OS. (a) The effect of PAGE4 overexpression with or without 400 µM H2O2 on apoptosis levels of WPMY-1 and PrPF cells was detected by flow cytometry. The lower right quadrant is early apoptosis, and the upper right quadrant is late apoptosis. (b) The apoptosis rate of each treatment group was statistically analyzed, and the calculated area of apoptosis rate was the Annexin V+/PI+ cell percentage, which was the sum of the percentage of cells in the lower right quadrant and the upper right quadrant. (c) The effect of PAGE4 overexpression with or without 400 µM H2O2 on the cell cycle of WPMY-1 and PrPF cells was detected by flow cytometry. Percentages of cell populations at different stages of the cell cycle were listed in the panel (%). (d) Histogram showing percentage of cell populations at different stages of the cell cycle (%). (e) Immunoblot assays showing the effects of PAGE4 overexpression with or without 400 µM H2O2 on the protein expression levels of CDK6, Cyclin D1, BAX, and Bcl-2 in WPMY-1 and PrPF cells. GAPDH is used as loading control. ns: no significant difference; *: p < 0.05; **: p < 0.01; ***: p < 0.001. ANOVA or Student's t-test.