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Fig. 5 | Molecular Cancer

Fig. 5

From: Tumor-suppressive circRHOBTB3 is excreted out of cells via exosome to sustain colorectal cancer cell fitness

Fig. 5

circRHOBTB3 is sorted into exosomes according to its specific motif sequence. (A) Observation of CRC cell exosomes by transmission electron microscopy (TEM) and Western blotting with anti-CD63 and anti-TSG101 antibodies. Anti-calnexin was used as a negative control, and cellular lysates were used as positive loading controls. (B) Exosomal secretion assay of normal cell lines, tumor cell lines and (C) RKO cells transfected with truncated circRHOBTB3 vector. (D) Schematic diagram of truncated circRHOBTB3 vector construction and primer design for exosomal secretion assay. (E) Exosomal secretion assay of RKO cells transfected with 25 nt truncated circRHOBTB3 vector. (F) The specific amino acid sequence of SNF8 shown by second-order mass spectrometry. (G) WB of RNA pull-down products confirmed the interaction of circRHOBTB3 with SNF8. (H) RT-qPCR of the anti-FLAG RIP assay. RHOBTB3 and circZDHHC21 were used as negative controls. (I) Expression of circRHOBTB3 in the cytosome and (J) exosomes of SNF8-KD HCT116 and RKO cells. (K) Western blot and (L) RT-qPCR of anti-SNF8 RIP assay in full-length and Δ141–240 circRHOBTB3 re-expressed SW480-KO cells. All experiments were repeated for three times, data were shown as mean ± SD (I, J) or mean ± SEM (B, C, E, H, L), * P < 0.05, ** P < 0.01, *** P < 0.001, NS P > 0.05, in Student’s test (I, J) or paired Student’s test (H, L)

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