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. 2017 Jul 3;216(7):2217–2230. doi: 10.1083/jcb.201601109

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© 2017 Stik et al.

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Figure 3. — Role of stromal EVs in HSPC maintenance ex vivo. (A) Proliferation of LSK cells cocultured 96 h with two quantities of stromal EVs in the absence of cytokines (n = 6). (B) Clonogenic potential of LSK cells cocultured 96 h with two quantities of stromal EVs (n = 6). (C) Representative micrographs depicting colony-forming units (CFUs). Note the difference in number (nb) and size between the two doses of AFT EVs. These figures are composites of multiple separate images. (D) Dose effect of AFT EVs on the maintenance of LSK cells after 96 h (n = 3). (E) Quantitative PCR measurement of RalB expression in AFT-shSCR– or AFT-shRalB–transduced cells. (F) Total protein quantification of EVs collected after culture of AFT-shSCR or AFT-shRalB cells. (G) Western blot analysis of CD63 protein expression in AFT-shSCR or AFT-shRalB cells. (H) Percentage of CD45⁺ cells after LSK/AFT-shSCR or AFT-shRalB cocultures during 96 h. (I) Representative flow cytometry analysis of CD45 expression in cocultures. (J) Clonogenic potential of LSK cocultures with AFT-shSCR or AFT-shRalB cells (n = 3). G, granulocyte; GEMM, granulocyte/erythrocyte/macrophage/megakaryocyte; GM, granulocyte/macrophage; M, macrophage. (K) Clonogenic potential of LSK cocultures with AFT cells or different fractions of AFT CM after sequential centrifugation at 300 g (CM AFT-300g), 2,000 g (CM AFT-2kg), and 110,000 g (CM AFT-110kg; n = 3). Error bars show SEM. *, P < 0.05.