Fig. 5

Detection of labelled mRNAs originating from bone-metastatic prostate cancer cell lines in recipient osteoblasts and the contribution to overall transcript abundance in recipient osteoblasts. a PNT1A (normal prostate), PC3, C4-2, C4-2-4B (prostate cancer) or hOB (osteoblast) cells were grown in the presence of 5EU to label nascent RNA transcripts. Post-labelling EVs produced from these cell lines were isolated and applied to hOBs cells grown under standard conditions (no EU label). After 48 h, the EV-treated hOB cells were lysed and the total RNA extracted, from the pool of total RNA EU-labelled RNA was precipitated and the presence of labelled CSF-1, VEGFA, MCP1, Runx2 and FGF2 quantified. b All EU-labelled transcripts were detected at significantly higher levels in hOB cells treated with EVs isolated from EU-labelled PC3 cells compared with EU-labelled PNT1A cells (CSF-1 p = 0.0395, VEGFA p = 0.0134, MCP1 p = 0.0086, Runx2 p = 0.0168, FGF2 p = 0.0284) (n = 3). Similar treatment with prostate cancer C4-2 and C42-B4 EVs, resulted in detection of increased 5EU-labelled CSF-1, MCP1 and RUNX2, but not VEGFA or FGF2 as seen with PC3. (p = 0.001, p = 0.001, p = 0.001 and p < 0.0001, p = 0.001, p = 0.002, respectively) (n = 2). c Analysis of total RNA extracted from hOBs after treatment with labelled PC3-EVs in a demonstrated a significant increase in abundance of CSF-1 and a smaller increase in FGF2 (similar results were obtained following treatment with C4-2 EVs Supplementary Figure 6). d To determine how the miRNA element of the EV cargo may influence the subsequent processing of the mRNA cargo component in the recipient cell, PC3 cells were transfected with siRNA against dicer (si-dicer) or a non-targeting siRNA (si-scr) control. 48 h post transfection EVs were isolated from the PC3 cells and transferred to hOB (osteoblast) cells growing under normal conditions. The abundance of VEGFA, MCP1 and Runx2, shown previously to have no altered abundance within the osteoblast transcriptome a–b, were quantified. A significant increase in the expression of MCP1 (p<0.0001) was determined, but no change in VEGFA and Runx2 could be detected (n = 3). Western blot confirmation of Dicer knockdown and controls for the siRNA loading into EVs is included in Supplementary Figure 4. t test with Holm–Sidak correction error bars represent standard deviation n = 3. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001