Fig 2: EphA2 interacts with the nuclear import machinery via a nuclear localization sequence in its cytotail.a Schematic diagram of the sequence of endosomally localized EphA2, indicating its predicted juxtamembrane NLS sequence and amino acid substitutions deployed to generate NLS1 and NLS2 mutants. b Schematic diagram of the EphA2-TurboID construct and workflow to determine the proximity interactome of EphA2. c H1299 cells stably expressing EphA2-TurboID constructs with wild-type EphA2 (WT) or mutated EphA2 NLSs (NLS1 and NLS2) were incubated with biotin for 1 h. Biotinylated proteins were isolated and analysed using MS-based proteomics (b). Scatter plots display the biotin-labelled protein enrichment (x-axis) commanded by wild-type EphA2 by comparison with EphA2s harbouring mutated NLS sequences (NLS1 and NLS2). Significance scores (−Log Student’s t-test p-value, n = 5 independent experiments) are plotted on the y-axes. Student’s t-test values > −Log 1.4 (

nuclear pore components that were identified in the proximity interactome. d Schematic diagram of the nuclear pore complex. Annotated proteins correspond to EphA2 proximity interactors confirmed in e. e Cells expressing EphA2-TurboID constructs were incubated with biotin in the presence or absence of HGF for 1 h. Biotinylated target proteins were analysed using western blotting. f Cells expressing the indicated GFP-tagged EphA constructs were incubated in the presence or absence of HGF for 5 min. EphA2-GFPs were immunoprecipitated and the presence of importins -α5 and -β1 was determined using western blotting. Blots are representative of four independent experiments. g Cells expressing EphA2-GFP (green) in combination with mCherry-importin-α5 (upper panels, red) or mCherry-importin-β1 (lower panels, red) were imaged by fluorescence time-lapse microscopy and movies collected (Supplementary Movies 2 and 3). Stills from these movies (selected at 10 s intervals) are displayed in the right panels. Scale bar, 10 μm. h Cells expressing the indicated GFP-tagged EphA2 constructs were treated with HGF for the indicated times and delivery of the tagged EphA2s to the nuclear fraction was determined using capture-ELISA as for Fig. 1d. Values are mean ± SEM, n = 5 independent experiments, statistical test is repeated-measures two-way ANOVA.

Fig 3: The overexpression of EphA2 increased tumorigenic cell growth of CC. A, Western blot analysis of EphA2 protein expression in C4‐i and HeLa cells transfected with EphA2 expression plasmid (C4‐i‐EphA2‐OE#1, C4‐i‐EphA2‐OE#2, HeLa‐EphA2‐OE#1, HeLa‐EphA2‐OE#2) and control vector (C4‐i‐Con, HeLa‐Con). B, MTT analysis for cell proliferation of C4‐i cells transfected with EphA2 expression plasmid (C4‐i‐EphA2‐OE#1, C4‐i‐EphA2‐OE#2, HeLa‐EphA2‐OE#1, HeLa‐EphA2‐OE#2) and control vector (C4‐i‐Con, HeLa‐Con). C, Transwell migration assay of C4‐i or Hela cells transfected with EphA2 expression plasmid (C4‐i‐EphA2‐OE#1, C4‐i‐EphA2‐OE#2, HeLa‐EphA2‐OE#1, HeLa‐EphA2‐OE#2) and control vector (C4‐i‐Con, HeLa‐Con). D, Wound‐healing assay of C4‐i cells transfected with EphA2 expression plasmid (C4‐i‐EphA2‐OE#1, C4‐i‐EphA2‐OE#2, HeLa‐EphA2‐OE#1, HeLa‐EphA2‐OE#2) and control vector (C4‐i‐Con, HeLa‐Con). E, Tumour sphere assay of C4‐i cells transfected with EphA2 expression plasmid (C4‐i‐EphA2‐OE#1, C4‐i‐EphA2‐OE#2, HeLa‐EphA2‐OE#1, HeLa‐EphA2‐OE#2) and control vector (C4‐i‐Con, HeLa‐Con). F, Xenograft tumour development in NOD SCID mice inoculated with control (C4‐i‐Con) or EphA2 overexpression (C4‐i‐EphA2‐OE) cells (n = 4). Four weeks later, mice were sacrificed, and tumours were collected (left) and the volume was calculated (right). G, The tumours weight was calculated from Figure 2F. The results in (B), (F), and (G) represent the mean ± SD. Statistically significant differences are indicated. **P < .01

Fig 4: YB1 induces sunitinib resistance by mediating EphA2 signaling in vitro.a GSEA analysis between YB1 and EphA2 mRNA levels and multiple-drug resistance signaling pathways. b Western blot analysis of YB1 and EphA2 protein expression in parental and sunitinib-resistant cells. The values indicate protein expression levels relative to actin levels. c CCK8 assay analysis for the effect of YB1 knockdown on the sensitivity of sunitinib-resistant cells to sunitinib treatment. d CCK8 assay analysis for the effect of EphA2 knockdown on the sensitivity of sunitinib-resistant cells to sunitinib treatment. e CCK8 assay analysis for the sensitivity to sunitinib treatment of 786-O-R and ACHN-R cells with YB1 overexpression/EphA2 knockdown or vector/EphA2 knockdown (***P < 0.001, **P < 0.01, *P < 0.05, compared with the corresponding control).

Fig 5: The EphA2 mRNA expression and its association with tumor stage using Bittner database of ONCOMINE database. The EphA2 mRNA expression and its association with presence or absence of distance metastasis using Bittner database of ONCOMINE database. The EphA2 protein expression and its association with patients’ outcome in our patients’ cohort. The EphA2 mRNA expression and its association with patient outcome presented as Overall Survival (OS) using KM plotter database in stage I renal cell carcinoma patients. The EphA2 mRNA expression and its association with patient outcome presented as OS using KM plotter database in stage III renal cell carcinoma patients. The relationship between EphA2 expression and Ki-67 expression. EphA2 = ephrin type-A receptor 2, mRNA = messenger ribose nucleic acid.