Fig 1: Effects of RNF126 and PTEN on BCa cell progression in vivo.A, B The qRT-PCR and western blotting showed the stable knockdown of RNF126 and PTEN mRNA and protein levels in T24 cells. C LV-control, LV-shRNF126, LV-shPTEN and LV-shRNF126 + shPTEN T24 cells were subcutaneously injected into four groups mice to generate the xenograft models. When the mice grew for 5 weeks, the xenografts were taken out and photographed (n = 8). D Tumor volume growth of BCa cells was measured twice a week until it grew for 5 weeks. E After the mice were sacrificed, the tumor weight was measured. F IHC demonstrated the expression of RNF126, PTEN and Ki67 proteins in tumors. Scale bar = 100 μm. G, H The mice’s tails were intravenous injected the LV-control, LV-shRNF126, LV-shPTEN and LV-shRNF126 + shPTEN T24 cells to establish the pulmonary metastasis models (n = 3). The BCa cell migration capacity was demonstrated by the fluorescence of pulmonary metastases. I After injection for 8 weeks, the mice were sacrificed and the lungs were taken out. Then, the lungs were stained with HE. The arrows denote the tumors transferred to the lungs. Scale bar = 100 μm. J The quantification of pulmonary metastases was determined by counting the metastatic nodules. ***p < 0.001, **p < 0.01, *p < 0.05.
Fig 2: Expression of RNF126 in BCa of TCGA samples and different cell lines.A RNF126 expression in the normal and primary tumor of the bladder. B RNF126 expression is based on different histological subtypes: papillary tumor and non-papillary tumor. C Expression of RNF126 in BCa based on the patient’s race. D Promoter methylation level of RNF126 in BCa. E RNF126 promoter methylation profile is based on histological subtypes. F The qRT-PCR analysis confirmed the transcription level of RNF126 in normal bladder epithelial cells SV-HUC-1 and various BCa cell lines (T24, UMUC3, 5637, BIU78). ***p < 0.001, **p < 0.01, *p < 0.05.
Fig 3: RNF126 deficiency affects the cell cycle and EGFR/PI3K/AKT signaling pathway in BCa cells.A, B The flow cytometry analysis demonstrated the percentage (%) of cells in different phases of cell cycle. The T24 and UMUC3 cells were treated with RNF126-si1 and si2 for two days. C, D The percentage (%) of cells in each phase was statistically analyzed from three independent experiments. E The western blotting revealed downregulation of protein associated with the cell cycle (CCND1, CDK2 and CDK4) in the BCa cells after silencing RNF126. The internal control was GAPDH. F The relative mRNA level of CCND1 in different cell types, treatment of siRNA. H The western blotting showed the protein level of EGFR, PI3K, total and phosphorylated AKT, mTOR, and PTEN in BCa cells treated with RNF126-si1and si2. The internal control was GAPDH. I The relative mRNA level of EGFR, PI3K, AKT, PTEN in different cell types, and siRNA treatment. ***p < 0.001, **p < 0.01, *p < 0.05.
Fig 4: Depletion of RNF126 suppressed BCa cell proliferation and metastasis through upregulating PTEN.A The MTT assay evaluated the growth and viability of the T24 cell from day 1 until day 5. A lentiviral shRNF126 and a lentiviral control group shNC stably transfected T24 cell were established. The cells were transfected with siNC (red line: shNC and orange line: shRNF126) or siPTEN (olive line: shNC + siPTEN and green line: shRNF126 + siPTEN). B, C The flow cytometry analysis demonstrated the distribution of cells in different phases of cell cycle. The four groups were the same as above. D The colony formation assay showed the effect of RNF126 knockdown on the cell survival of UMUC3 and T24 cells after the indicated transfection. Scale bar = 1 cm. E Colony numbers were counted and plotted as indicated. F The transwell assay evaluated cell migration of the RNF126 or PTEN knockdown treated T24 cells. Scale bar = 100 µm. G The relative number of cell migration was statistically analyzed. H The western blotting indicated the protein level of RNF126, PTEN, PI3K, p-AKT, AKT, CCND1, p21, and N-Cad. Loading control was GAPDH. ***p < 0.001, **p < 0.01, *p < 0.05.
Fig 5: RNF126 interacts with PTEN.A The co-immunoprecipitation of GFP-PTEN and FLAG-RNF126 in 293T cells. The 293T cells were transfected with FLAG-RNF126 and GFP-PTEN plasmids for 36 h. B The endogenous co-immunoprecipitation of PTEN and RNF126 in T24 cells. C–E The interactions between full-length RNF126 and truncated forms of GFP-PTEN were observed by co-immunoprecipitation in 293T cells. F The interactions between full-length PTEN and truncated forms of FLAG-RNF126 were observed by co-immunoprecipitation in 293T cells. G Protein domains of RNF126 and PTEN. Numbers indicate how long the proteins were cut off. RNF126 protein was truncated between the RING finger domain and the Zn finger domain. PTEN protein was truncated to reserve the C2 domain.
Supplier Page from Abcam for Anti-RNF126 antibody