Fig 1: PRDM4 inhibited the tumor formation of cervical cancer cells in vivo.The expression of PRDM4 protein in cervical cancer cell lines was detected by IHC staining (A) and Western blots (B). C PRDM4 protein was detected in the PRDM4-overexpressing or PRDM4-silenced HeLa and SiHa cells. D, E The tumor growth curve and weight were analyzed in the PRDM4-overexpressing or PRDM4-silenced HeLa and SiHa cells with six female nude mice in triplicate. F, G Tumor-free survival was analyzed in the PRDM4-overexpressing or PRDM4-silenced HeLa and SiHa cells. Scale bar: 50 µm. *p < 0.05 and **p < 0.01.
Fig 2: PRDM4 inhibited cell proliferation by regulating cell cycle-related proteins.A The differential mRNA expression in HeLa-PRDM4 and HeLa-GFP cells determined by RNA-seq showed 1664 upregulated genes and 510 downregulated genes. B Cell cycle-related gene levels between the HeLa-PRDM4 and HeLa-GFP cells. C The transcript levels of p53, p21, p27, Cyclin D1, and CDK4 were detected in the PRDM4-overexpressing and PRDM4-silenced cells by real-time PCR. D–G Cell cycle proteins were detected by Western blotting, analyzed by utilizing a protein imprinting imaging system (Tanon 5200, China) and expressed as relative expression after normalization to GAPDH. *p < 0.05 and **p < 0.01, while ns indicates no significant difference.
Fig 3: PRDM4 inhibited cell proliferation by blocking the cell cycle transition from G0/G1 to S phase.A Cell viability was determined in the PRDM4-overexpressing or PRDM4-silenced HeLa and SiHa cells by MTT assays. B Cell proliferation assays were performed in the PRDM4-overexpressing or PRDM4-silenced HeLa and SiHa cells. C, D The expression levels of PRDM4 and Ki67 in the tumor tissues are shown. E, F Cell cycle distribution was analyzed by FACS in the PRDM4-overexpressing or PRDM4-silenced cells. *p < 0.05 and **p < 0.01.
Fig 4: PRDM4 inhibited the activity of the PI3K/AKT pathway by transactivating PTEN.A, B Bioinformatics analysis of RNA-seq was performed to confirm that the PI3K/AKT pathway and target genes were significantly enriched in HeLa-PRDM4 cells. C The mRNA level of PTEN was detected by real-time PCR. D, E The expression levels of AKT, p-AKT, and PTEN were detected by Western blots. F The PRDM4 DNA-binding motif containing TC/AATTA was predicted by the JASPAR database (http://jaspar.genereg.net/), and the diagram of the PTEN promoter containing the possible cis-acting elements bound by PRDM4 is also shown. G Luciferase activity of PTEN promoter deletions relative to Renilla activity was detected in the PRDM4-overexpressing or PRDM4-silenced cells and the controls. H The luciferase activity of the PTEN promoter P3 mutations was detected in the PRDM4-overexpressing HeLa and SiHa cells. I The qChIP assay results are shown for the PRDM4-overexpressing and PRDM4-silenced HeLa and SiHa cells. Statistical significance is denoted by the symbols *p < 0.05, **p < 0.01), ***p < 0.001, while ns indicates no significant difference.
Fig 5: Silencing the expression of PTEN rescued the cell growth potential induced by PRDM4 overexpression.A The expression levels of PTEN, AKT, p-AKT, p21, and p27 were detected by Western blots in the PTEN-silenced HeLa-PRDM4 and SiHa-PRDM4 cells. B, C PTEN was silenced by specific shRNA targeting the PTEN CDS region in the PRDM4-overexpressing HeLa and SiHa cells, and the associated tumor weights and growth curves are shown. D The expression levels of AKT and p-AKT were detected in the HeLa-PRDM4 and SiHa-PRDM4 cells treated with the PTEN inhibitor SF1670. E The cell proliferation and cell vitality were assessed in the HeLa-PRDM4 and SiHa-PRDM4 cells treated with the PTEN inhibitor SF1670. F The correlation between the PRDM4 and PTEN mRNA levels in cervical cancer was analyzed using the TCGA database (R = 0.24, p < 0.05). G A proposed model of the PRDM4-mediated enhancement of PTEN inactivation of the PI3K/AKT pathway in cervical cancer progression. The data are presented as the mean ± SD of experiments in triplicate. Statistical significance is denoted by the symbols *p < 0.05, **p < 0.01, ***p < 0.001.
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