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. Author manuscript; available in PMC: 2017 Jan 15.
Published in final edited form as: Cancer Res. 2015 Dec 16;76(2):440–452. doi: 10.1158/0008-5472.CAN-15-1640-T

Figure 3. Persistent TORC1/2 inhibition drives Notch1 signaling and a CSC-like phenotype.

Figure 3

(A) SUM159 and (B) BT549 cells were treated with progressively higher concentrations (5–300 nM) of BEZ235 (BEZR) and MLN128 (MLNR) over a period of 8 weeks. FACS analysis for the CSC markers were quantified (SUM159: *P=0.0002, **P=0.002; BT549: *P<0.03).

(C) SUM159 and (D) BT549 cells were treated as described in (A,B) and seeded as mammospheres; mammosphere number was determined after 7 days using GelCount (SUM159:*P<0.01; BT549:*P<0.02). Two representative images of mammospheres from each treatment are shown (magnification = 40×).

(E) Immunoblot analysis of SUM159 and BT549 BEZR and MLNR cells was performed using the indicated antibodies.

(F) SUM159 BEZR and MLNR cells were transfected with the HES1-Firefly Luciferase and CMV-Renilla constructs. The Dual Luciferase Assay was performed after 48 h as described in Experimental Procedures (*P<0.0001, **P=0.03).

(G) SUM159 CTL, BEZR and MLNR cells were cultured in the continuous presence of vehicle or their respective inhibitor or taken off drug selection for 72 h, followed by immunoblot analysis for NICD, C-MYC and JAG1.

Error bars represent mean ± SEM.