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. Author manuscript; available in PMC: 2015 Oct 23.
Published in final edited form as: Cell. 2014 Oct 23;159(3):499–513. doi: 10.1016/j.cell.2014.09.051

Figure 3. Stromal exosomes are regulated by RAB27B and transfer 5’-triphosphate RNA to activate RIG-I in breast cancer cells.

Figure 3

A) Exosomes were isolated from mono-culture of MRC5 fibroblasts (Stroma) or 1833 IRDS-R (right) or from co-culture (left) and profiled by antibody array for the indicated exosome markers. GM130 is a check for cellular contamination. Positive (+) and negative (−) controls are labeled. B) Averaged microarray expression of the indicated RABs from MRC5 in mono-culture (Stroma) or after co-culture with IRDS-R or IRDS-NR are shown as a heat map. Immunoblot (right) for Rab27b protein expression in MRC5 after co-culture with MDA-MB-157 or 1833 IRDS-R (Figure S3A) compared to MRC5 mono-culture. C) IRDS expression in 1833 IRDS-R after addition of CM isolated from co-culture using MRC5 transfected with siRAB27B compared to siControl (n=3). D) Exosome transfer to 1833 IRDS-R after co-culture with or without RAB27B knockdown (left) or addition of co-culture CM cleared of debris and apoptotic bodies (right). E) Average IRDS gene expression (mean expression of IFIT1, MX1, and STAT1) in response to exosomes (Exo, n=5) or co-culture CM (n=6) plotted against RIG-I levels after knockdown in 1833 IRDS-R. F) IRDS gene expression from two representative data points used to generate plot in Figure 3E are shown relative to siControl. G) IRDS gene expression after RNA from exosomes (ExoRNA), cellular RNA, or a positive control HCV RNA was transfected into 1833 IRDS-R with or without RIG-I knockdown (n=4). IFI16 is a non-IRDS gene used as a negative control. H) Expression of IRDS genes IFIT1 and MX1 resulting from transfection of ExoRNA after RNase treatment, or I) removal of 5’-monophosphate (5’-p) and/or 5’-triphosphate (5’-ppp) (n=3). An in vitro transcribed 5’-ppp RNA (IVT5’ppp) is used as a positive control. Shown are RNA motifs remaining after enzyme modification with alkaline phosphatase (AlkPase), Terminator exonuclease (Term), and tobacco acid pyrophosphatase (TAP). IVT5’ppp serves as a control for RNA enzyme modification by AlkPase and TAP. J) Distribution of known gene transcripts and intergenic transcripts from rRNA-depleted exoRNA and cellular RNA from 1833 IRDS-R co-culture (left). Distribution of major repetitive elements and transposable element classes for intergenic transcripts are shown on right. K) ExoRNA enrichment for major subfamilies of transposable elements and satellite sequences compared to cellular RNA. *p < 0.05. See Figure S3.