ROME drives intravasation through vimentin. A, Timeline of zebrafish xenograft experiments. B, Zebrafish xenograft experiments with three different ROME-overexpressing Ewing sarcoma cell lines. Statistical significance was determined via the Fisher exact test (two-sided). C, Zebrafish xenograft experiments comparing WT and ROME-KO TC-71 cells. Statistical significance was determined via the Fisher exact test (one-sided). D, ROME and vimentin proteins co-immunoprecipitate in three different Ewing sarcoma cell lines. For TC-32 ROME blot, line denotes higher exposure on left to visualize input and lower exposure on right. E, SPR sensorgrams showing direct binding between the recombinant full-length ROME protein and vimentin protein. Vimentin was immobilized on the surface and full-length ROME protein was injected in duplicate at concentrations of 2,500, 800, 280, 90, 30, and 10 nmol/L. The red lines are the actual data, and the black lines indicate the curve fit. F, ROME expression reduces vimentin phosphorylation at serine 56 (S56) in a tet-inducible ROME-HA expression system (DOX = doxycycline 250 ng/mL). Densitometry analysis of Western blot bands is shown in the bottom right corner (n = 4 independent experiments). G, Western blotting was used to confirm vimentin knockdown by siRNA in STA-ET-7.2 cells with EV or ROME overexpression. H, Vimentin knockdown selectively reduced ROME-driven intravasation in ROME-overexpressing STA-ET-7.2 cells compared with an EV control [n = 102 (EV + siNT), 116 (ROME + siNT), 88 (EV + siVimentin), and 119 (ROME + siVimentin)]. I, ROME mRNA expression positively correlates with vimentin mRNA expression in analysis of 13,313 patient samples. [A, Created in BioRender. Lab, S. (2026) https://BioRender.com/fp8c676.]
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