Fig 1: Expression of PAGE4 when exposed to H2O2 in WPMY-1 and PrPF cells. (a) The blank control group and 100, 200, 400, and 800 µM H2O2 treatment groups were set, respectively. The cell proliferation activity of WPMY-1 and PrPF cells was determined by CCK-8 assay. The IC50 value was 431.9 µM and 409.2 µM, respectively. The mRNA expression of (b) SOD1 and (c) PAGE4 in WPMY-1 and PrPF cells, which were treated with 400 µM H2O2 combined with or without 5 mM NAC. (d) Immunoblot assays demonstrated the protein expression of SOD1 and PAGE4 in WPMY-1 and PrPF cells, which were treated with 400 µM H2O2 combined with or without 5 mM NAC. (e) Immunofluorescence was used to detect the expression of PAGE4 in WPMY-1 cells without or with 400 µM H2O2 treatment. Left: DAPI (blue) shows nuclear staining; middle: Cy3 immunofluorescence (red) shows PAGE4 protein; right: merged image. Representative graphs were selected into figure. GAPDH is used as loading control. ns: no significant difference; **: p < 0.01; ***: p < 0.001. ANOVA or Student's t-test. The scale bars are 100 µm.
Fig 2: 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 3: 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 4: The effect of overexpression of PAGE4 on cell proliferation and oxidative stress level of WPMY-1 and PrPF cells under OS. (a) qRT-PCR was used to verify the efficiency of PAGE4 overexpression in WPMY-1 and PrPF cells at the transcriptional level. (b) Immunoblot assays showing the efficiency of PAGE4 overexpression in WPMY-1 and PrPF cells at the protein level. (c) The cell viability of WPMY-1 (i) and PrPF (ii) after overexpression of PAGE4 with or without 400 µM H2O2 treatment at different time points by CCK-8 assay (*: vector vs. vector + H2O2; #: vector + H2O2 vs. PAGE4 + H2O2). qRT-PCR was used to verify the effect of PAGE4 and combined or noncombined 400 µM H2O2 on the transcription level of (d) SOD1 and (e) CAT in WPMY-1 and PrPF cells after PAGE4 overexpression. (f) Immunoblot assays showing the effects of PAGE4 overexpression with or without 400 µM H2O2 on the protein expression levels of SOD1 and CAT in WPMY-1 and PrPF cells. (g) Statistical analysis of mean fluorescence intensity (MFI) of DCFH-DA in WPMY-1 and PrPF cells after different treatments. (h) DCFH-DA fluorescent probe was used to analyze the accumulation of ROS by PAGE4 overexpression in WPMY-1 and PrPF cells under OS, and different color curves were used to represent different treatment groups. 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.
Fig 5: The effects of overexpression of PAGE4 on MAPK signaling pathways of WPMY-1 and PrPF cells under OS. (a) Immunoblot assays showing the effects of PAGE4 overexpression 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; *: p < 0.05; **: p < 0.01. ANOVA or Student's t-test.
Supplier Page from Abcam for Anti-PAGE4 antibody