Fig 1: RNF25 is overexpressed in human HCC tissue and associated with poor prognosis. A) RNF25 protein levels in patients with indicated cancer types according to The Human Protein Atlas dataset. HPA036420 and HPA036421 represent two distinct antibodies recognizing RNF25 protein. B–F) The correlation between RNF25 mRNA level and ECAD protein level in different cancer types was evaluated using the TCGA dataset. G) Immunohistochemistry (IHC) assays showing the protein level of RNF25 in HCC patients with or without metastasis. Scale bars, 40 µm (zoom out) or 10 µm (zoom in). H) HCC patients in (G) were grouped according to their tumor grade followed by evaluation of RNF25 expression. I) HCC patients in (G) were grouped according to their RNF25 expression level followed by an analysis of their overall survival. J) The correlation of protein levels of RNF25 and ECAD in clinical HCC samples was determined by IHC. Scale bars, 40 µm (zoom out) or 10 µm (zoom in). p‐value was determined by Pearson correlation test (B–F,J), unpaired t‐test (G), one‐way ANOVA (H), and Log‐rank (Mantel‐Cox) test (I). * p < 0.05, ** p < 0.01; *** p < 0.001.
Fig 2: Redox modification of Protein Kinase A at C200/344 upregulates kinase activity of PKA. A) The band shift was detected in non‐reduced SDS‐PAGE using HEK293T cells expressing wild‐type or cysteine‐mutated PRKACB. The cells were treated with 100 µm H2O2 for 1 h before harvesting. B) Activity of wild‐type or cysteine mutated PRKACB was measured with or without the treatment of 100 µm H2O2 for 1 h. C) The phosphorylation of RNF25 by wild‐type or cysteine‐mutated PRKACB was determined with immunoprecipitation assays using HEK293T cells. D) Co‐IP assay showing the ubiquitination of ECAD in HCC cells expressing wild‐type or C200S/C344S mutated PRKACB. E,F) Immunoblotting and qPCR assays showing the protein expression and mRNA levels of ECAD in HCC cells expressing wild‐type or C200S/C344S mutated PRKACB. G) Transwell assay showing the migration and invasion of HCC cells with or without the mutation of PRKACB and the ectopic expression of S450D RNF25. Data are mean ± SD from at least three independent repeats. P‐value was determined by unpaired t‐test (B,F,G). * p < 0.05, ** p < 0.01; *** p < 0.001. NS, not significant.
Fig 3: The effects of altered RNF25 expression on the anti-tumor efficacy of gefitinib in vivo.a, b Mice bearing H1650/shRNF25 or H1650/shControl tumor xenografts were injected twice a week with 50 mg/kg gefitinib or vehicle by i.p., and tumor growth was monitored for 45 days. Photographs of tumors resected from each group at the end of experiments are shown (a) n = 3/group. Profiles of tumor growth during the 45 days of gefitinib treatment are also shown; For each treatment group, data are presented as mean tumor volume (mm3) ± SEM (b). c The levels of protein expression in the resected tumors were analyzed by western blotting. β-Actin was used as a loading control
Fig 4: RNF25 knockdown increases gefitinib sensitivity by inhibiting ERK reactivation in gefitinib-resistant cells.a H1650 cells were transfected with siRNAs for RNF25 individually or as a mixture, and then treated with gefitinib (5 μM) or DMSO for 72 h. Cell proliferation was measured by cell counting. Values are mean ± SD of three independent experiments. b, c H1650 cells stably transduced with a lentiviral vector carrying shRNF25 (H1650/shRNF25) or pLKO (H1650/shControl) were treated with gefitinib or DMSO for 72 h. The gefitinib sensitivity of each cell line was determined using varying concentrations of gefitinib, and the values are mean ± SD of two independent experiments (b) For reconstitution of RNF25 expression, H1650/shRNF25 (c). d HCC827 or PC-9 cells were transfected with pcDNA-RNF25 or a control vector for 24 h, and then treated with gefitinib (5 μM for H1650, 0.002 μM for HCC827, 0.05 μM for PC-9). For (c, d), values are mean ± SD of three independent experiments. e, f Phosphorylation levels of pro-survival pathway members were analyzed by western blotting in H1650 cells stably transduced with a lentiviral vector carrying shRNF25 or pLKO. The cells were treated with gefitinib (5 μM), ERK inhibitor (ERKi; SCH772984, 10 μM), and/or DMSO for 72 h before the western blot analysis. Values in the bar graphs are mean ± SD of three independent experiments. β-Actin was used as a loading control. g H1650 cells were treated with ERK inhibitor (10 μM) or vehicle in combination with varying concentrations of gefitinib. Values are mean ± SD of two independent experiments. Statistical significance was determined by the Student’s t-test (*p < 0.05 and **p < 0.01)
Fig 5: Phosphorylated RNF25 at Ser450 initiates E‐cadherin protein degradation. A) Functional score of phosphosites in RNF25 (gray) and human phosphoproteome (blue) according to a human phosphoproteome dataset. Mean, average score for all human phosphosites. Regulatory, phosphosites with known regulatory functions. Disease, phosphosites related to human diseases. B) Immunoprecipitation assays showing the phosphorylation of RNF25 in HCC cell lysate treated with or without lambda protein phosphatase. C) Immunoprecipitation assays showing the phosphorylation of wild‐type and S450A mutated RNF25 in PLC/PRF/5 stable cells. D) Western blot showing the ECAD protein levels in indicated stable cells. E) In vitro ubiquitination assay showing the ubiquitination of ECAD by wild‐type or S450D mutated RNF25. F) Immunoprecipitation assays showing the phosphorylation of RNF25 in indicated cells with or without the treatment of 100 µm H2O2 or 1 mm NAC for 1 h. G) Immunoprecipitation assays showing the ubiquitination of ECAD in indicated cells with or without the treatment of 100 µm H2O2 or 1 mm NAC for 24 h. H) Wound healing assay showing the migration of PLC‐RNF25‐WT, PLC‐RNF25‐S450A, and PLC‐RNF25‐S450D cells with or without the overexpression of ECAD or the treatment of 100 µm H2O2. Scale bars, 200 µm. I–K) Transwell assay showing the migration and invasion of indicated cells treated as (H). Scale bars, 200 µm. Data are mean ± SD from at least three independent repeats. P‐value was determined by an unpaired t‐test (J,K). * p < 0.05, ** p < 0.01; *** p < 0.001.
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