Supplementary MaterialsSupplementary file1 41598_2020_72503_MOESM1_ESM. protein and as a regulator of sEV secretion. Our data support a model whereby Rab13 can mediate its effects on cell proliferation and invasiveness via autocrine and paracrine signaling. no significance. To test the functional significance of Rab13-dependent secretion of sEVs, we utilized two unique Transwell assays (Supplementary Fig. S3a). We first tested whether parental DKO-1 cells, which express normal levels of Rab13, would impact the proliferation of DKs-8 cells in a co-culture environment. When plated on reverse sides of a Transwell membrane, the presence of DKO-1 cells in the donor compartment increased the proliferation of recipient DKs-8 cells when compared to control conditions with no donor cells (Fig.?1C). However, Rab13 knockdown in the donor DKO-1 cells abrogated the effect on proliferation in DKs-8 recipient cells following co-culture (Fig.?1C). Knockdown of Rab13 in KRAS wildtype DKs-8 donor cells did not impact recipient cell proliferation (Supplementary Fig. S3b). This suggests that Rab13 regulates extracellular secretion in KRAS mutant CRC cells and that decreased levels of Rab13 blocks proliferation inducing effects in recipient cells. Previously, we showed that can undergo functional transfer from donor mutant KRAS cells to recipient wild type KRAS cells using luciferase reporter assays25. When we tested whether Rab13 knockdown might alter transfer, we again found that decreased levels of Rab13 reduced extracellular transfer of from DKO-1 donor to DKs-8 recipient cells resulting in increased luciferase reporter expression (Fig.?1D). Rab13 regulates anchorage-independent growth via sEVs sEVs from mutant KRAS cells can promote proliferation and anchorage-independent growth in wild type KRAS cells9,26. Thus, we tested whether Rab13 knockdown in DKO-1 cells would alter colony formation in soft agar assays. First, DKO-1 cells were incubated with DKO-1 sEVs prior to embedding in soft agar. Exposure of DKO-1 cells to sEVs in this manner increased the total quantity of colonies in soft agar, compared to untreated DKO-1 cells, consistent with previous results9 (Fig.?1E,F). We then tested the effects of Rab13 knockdown on colony growth in soft agar. Loss of Rab13 slightly reduced the number of colonies in soft agar (Fig.?1F). However, exposure of the knockdown lines to sEVs from normal DKO-1 cells restored the colony counts back to control DKO-1 levels (Fig.?1F). Besides growth in soft agar, we also found that sEVs from DKO-1 cells caused an increase in tumor-like, migratory colonies when produced in type-1 collagen (Supplementary Fig. S4). Again, knockdown of Rab13 blocked this effect, whereas exposure to sEVs from DKO-1 cells rescued migratory colony figures (Supplementary Fig. S4). Together, these results suggest that Rab13 regulates anchorage-independent and 3D growth through regulation of sEV secretion, linking sEV biogenesis and tumorigenesis in colorectal malignancy. Although knockdown of Rab13 caused an 80C90% decrease in AZD-9291 (Osimertinib) particle counts/cell (Fig.?1B), we nevertheless sought to test whether EVs from knockdown cells could rescue colony counts as in Fig.?1E,F. For this, we scaled up to generate equivalent amounts of vesicle preparations from knockdown cells and uncovered DKO-1 cells to those EVs. As shown in Supplementary Fig. S5, we found that EVs from Rab13 knockdown cells could increase the total number of colonies in DKO-1 cells and rescue colony growth in knockdown cells in soft agar. This indicates that even though Rab13 knockdown dramatically reduces EV release, the producing EVs still contain cargo that can AZD-9291 (Osimertinib) promote cell proliferation. Rab13 regulates the secretion of sEV markers To further investigate the role that Rab13 plays in vesicle secretion, sEVs were isolated from control and knockdown cells (Fig.?2A). Consistent with the overall decrease in sEVs observed after knockdown of Rab13, Rabbit polyclonal to ZNF561 we observed reduced levels of the classical exosome markers CD63, CD81, and TSG101 (Fig.?2B). Previous work has shown that 1-integrin can be detected in EVs27,28 and that Rab13 can regulate 1-integrin recycling in an epithelial malignancy model17. Furthermore, AZD-9291 (Osimertinib) 1-integrin was detected in non-exosomal EVs8. Thus, we tested whether knockdown of Rab13 would reduce secretion of AZD-9291 (Osimertinib) 1-integrin. As shown in Fig.?2A,B, knockdown of Rab13 reduced the levels of secreted 1-integrin. Open in a separate window Physique 2 Rab13 regulates and co-localizes with sEV markers. (A) AZD-9291 (Osimertinib) Cell lysates prepared from DKO-1 cells expressing an.