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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 2. Stromal cell interaction increases exosomes that upregulate ISGs through a RIG-I anti-viral pathway.

Figure 2

A) Importance scores (higher is more predictive) of PRRs from a multivariable random forest (RF) regression model to predict induction of IRDS after MRC5 co-culture with IRDS-Rs. The model explains 60.8% of the total variance. Adjusted effect of RIG-I on IRDS metagene expression is shown on right (red dashes are ± two SE). B) Expression of IRDS genes after siRNA to RIG-I (top row) or MYD88 (bottom row) in 1833 IRDS-R. Shown is a representative experiment (n=3). C) Cell death of 1833 IRDS-R after RT (n=4) and a representative BLI-based survival assay (n=2) after the indicated knockdown (RT on day 0). Photon flux (×106) for each well is shown. The control is same as Fig. 1J. D) Expression of IRDS genes in 1833 IRDS-R (middle) or MCF7 IRDS-NR (right) after addition of conditioned media (CM) from MRC5 fibroblasts (Stroma), IRDS-R or IRDS-NR (BrCa), or MRC5 co-culture with IRDS-Rs or IRDS-NRs (Co-cx). See schematic (left). E) CM collected after 48 hrs or the soluble fraction from CM (Soluble) was applied to 1833 IRDS-R and expression of IRDS genes was examined (n=4). F) Fold induction of IRDS genes in 1833 IRDS-R after addition of co-culture CM or purified exosomes (n=5). G) NanoSight quantification of exosomes (left) from 1833 IRDS-R, MRC5 fibroblasts (Stroma), and MRC5 co-culture using either 1833 IRDS-R or IRDS-NR (MDA-MB-468 or MCF7). Immunoblot for TSG101 (right) using 1833 IRDS-NR and MDA-MB-468 IRDS-NR. H) MRC5 fibroblasts (Stroma) or 1833 IRDS-R were labeled with green or red lipophilic dye in mono-culture (left and middle). For co-culture (right), MRC5 (arrows) were labeled red and breast cancer cells green. Scale bar is 40 microns. I) Representative flow cytometry of DiD dye transfer from MRC5 stroma to 1833 IRDS-R or MDA-MB-468 IRDS-NR. J) Exosome transfer from co-culture after TSG101 knockdown (left) and after addition of the co-culture CM cleared of debris and apoptotic bodies (right) (n=4). K) IRDS gene induction by co-culture CM after TSG101 knockdown in 1833 IRDS-R, MRC5 stroma, or both (n=3). Gene expression and significance levels are relative to siControl. *p < 0.05. See Figure S2.