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. Author manuscript; available in PMC: 2019 Jul 5.
Published in final edited form as: Cell. 2017 Jul 13;170(2):352–366.e13. doi: 10.1016/j.cell.2017.06.031

Figure 5. NOTCH1-MYC Signaling Couples Stromal Activation with Unshielding of POL3-Driven Stromal RN7SL1.

Figure 5.

(A) Gene expression in MRC5 stromal cells (panels 1 and 3), ISG-R 1833 breast cancer cells (panel 2), or ISG-NR breast cancer cells (panel 4) after mono-culture versus co-culture. Genes significantly upregulated after co-culture are indicated by blue dots. Cancer-associated ISGs are marked red.

(B) RN7SL1 levels in MRC5 stromal cells after co-culture with ISG-R or ISG-NR breast cancer cells.

(C) Immunoblot of NOTCH family members ortheiractivation domain (NICD1, NICD3) in MRC5 stromal or ISG-R 1833 breast cancer cells. Activation of NOTCHI/ NOTCH3 is indicated by appearance of NICD1/NICD3 in absence of GSI, while activation of NOTCH2 is indicated by accumulation of cleavage product (arrow) with GSI.

(D) Immunofluorescence and quantitation of MYC+ nuclei in MRC5 stromal or 1833 breast cancer cells with or without GSI.

(E) SRP9 and SRP14 protein levels in MRC5 stromal cells after mono-culture or after ISG-R or ISG-NR co-culture.

(F and G) Immunoblot for the indicated proteins with quantification of biological replicates(right) (F), or(G) cellular RN7SL1 expression in MYC-ER MEFs treated with vehicle control (CTL) or 4OHT (+MYC).

(H-K) RBP-shielding of exoRNA isolated from MYC-ER MEFs (H), or (I) ISG expression in ISG-R breast cancer cells after addition of MYC-ER MEF exosomes. MYC-ER MEFs were treated with vehicle control (CTL) or 4OHT (+MYC) with or without POL3 inhibitor (POL3i) (n = 3). TSG101 is a non-ISG not expected to change. Experiments in (H) and (I) were repeated in (J) and (K) but with overexpression of SRP9/14 (SRP) or empty vector (EV) (n = 3).

(L) RBP-shielding of exoRNA isolated from ISG-R co-cultures using MRC5 cells treated with control (CTL) or MYC siRNA(n = 3). Inset: stromal MYC protein after knockdown.

(M) ISG expression in 1833 breast cancer cells after addition of conditioned media from ISG-R co-cultures using siRNA targeting MYC (siMYC)-treated MRC5 cells (n = 3).

Error bars are SEM of biological replicates and *p < 0.05, **p < 0.01. See also Figure S5.