Fig 1: ORP5 is overexpressed in RCC cells and tumor tissues, and associated with tumor progression.A The expressions of ORP5 in several human RCC cell lines (ACHN, 786-O, and Caki-1) and human normal tubular epithelial cell line (HK-2) were measured by Western blot. Quantification of the ORP5 expression (right). B IHC assay of ORP5 protein expressions in para-cancer and cancer tissues in ccRCC patients. Scale bar, 50 µm (left) and 25 µm (right). C Representative images of IHC staining of ORP5 in RCC tissues. Scale bar, 50 µm. Error bars represent SD. **P < 0.01; ***P < 0.001.
Fig 2: ORP5 overexpression suppresses lysosomes degradation of c-Met.A Q-PCR analysis was carried out to examine c-Met mRNA levels after ORP5 overexpression and knockdown in RCC cells. B Western blot was carried out to assess the effects of overexpressing ORP5 on the half-life of c-Met in 786-O, ACHN cells treated with cycloheximide (CHX) at 20 µg/ml and collected at 0, 6, 12, and 24 h. C Western blot of c-Met expressions in the indicated cells treated with or without Chloroquine (100 µmol). D Immunoprecipitation and immunoblotting were performed to analyze the ubiquitination of c-Met when co-transfected with Ub and ORP5 expression plasmids in these two RCC cells. Error bars represent SD. *P < 0.05; **P < 0.01.
Fig 3: ORP5 promotes migratory and invasive capacities of RCC cells.A The protein levels of Flag and ORP5 in RCC cells with ORP5 knockdown or ORP5 overexpression were examined by western blot analysis. B The effect of ORP5 on cell motility was assessed by testing wound closure after upregulation or knockdown of ORP5 in RCC cells. Original magnifications, ×100 for (B). C Transwell assays were used to detect the changes in the migratory and invasive capacities of 786-O and ACHN cells after overexpression or downregulation of ORP5. Original magnifications, ×200 for (C). D The protein expressions of ORP5, N-cadherin, E-cadherin, Vimentin and Fibronectin in 786-O and ACHN cells after specific treatment were tested by Western blot. Error bars represent SD. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig 4: The graphic illustration of ORP5 modulation of c-Met expression and metastasis and invasion of RCC.ORP5 stabilizes the c-Met protein levels by promoting the ubiquitination of c-Cbl and subsequently inhibiting c-Met ubiquitination and lysosomal degradation.
Fig 5: ORP5 facilitates metastasis of RCC in vivo through upregulating c-Met expression.A 2 × 106 786-O and ACHN cells transfected with LV-shORP5 and LV-shNC were injected into the tail vein of randomly grouped nude mice, respectively (n = 5). At 35 days after injection, the nude mice were sacrificed, then the lungs were removed and photographed. The white arrows pointed to metastatic nodules. B The number of metastatic nodules in the lungs of each mouse was counted. C Weights of the lungs were recorded. D H&E staining was performed on lung sections. Scale bar, 200 μm. E Representative pictures of IHC staining of ORP5, c-Met in mice lung tissues. Scale bar, 25 μm. F Representative IHC images of ORP5 and c-Met in ccRCC tissues. Scale bar, 50 μm. G The relationship between ORP5 IHC score or c-Met IHC score in ccRCC tissues was analyzed by Pearson correlation analysis. Error bars represent SD. *P < 0.01; ***P < 0.001.
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