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. 2015 Sep 17;12(12):1968–1977. doi: 10.1016/j.celrep.2015.08.050

Figure 2.

Figure 2

Tamoxifen or Fulvestrant Treatment Upregulates Notch Target Genes in Patient-Derived Samples and PDXs

JAG1-NOTCH4 receptor signaling in ALDH-positive cells drives Notch activity in endocrine-resistant BC.

(A and B) Expression of Notch target genes HEY1 and HES1 was assessed by real-time qPCR analysis and compared to control to determine fold change. (A) Metastatic BC patient-derived cells were treated for 7–9 days with ethanol (control), tamoxifen (10−6 M), or fulvestrant (10−7 M) and a correlation between fold change of expression of HEY1 and HES1 and fold change of percentage of ALDH-positive cells is shown. (B) Early (HBCx34) and metastatic (BB3RC31) BC PDXs: the effect of in vivo treatment for 14 days with tamoxifen (10 mg/kg/day, oral gavage) or fulvestrant (200 mg/kg/week, subcutaneous injection) on HEY1 and HES1.

(C) NOTCH4, HES1, and JAG1 protein expression levels determined by western blot in metastatic (Met) (BB3RC31) BC PDX. β-actin was used as a reference for the loading control.

(D) NOTCH4, HES1, and JAG1 protein expression levels were determined by western blot in MCF-7 ALDH-negative and ALDH-positive sorted cells. MCF-7 cells were treated with tamoxifen or fulvestrant for 6 days before ALDH sorting.

(E) Wild-type MCF-7 cells (filled bars) and a CRISPR clone containing a disruption of NOTCH4 exon 2 (N4EX2 cells, hatched bars) treated in adherence with ethanol (Control, gray bars), 10−6 M tamoxifen (red bars), and 10−7 M fulvestrant (blue bars) for 6 days. N4EX2 cells’ fold change of MFE and ALDH-positive cells after treatments was compared to that of the wild-type cells.

(F and G) NICD4 and JAG1 rescue tamoxifen- or fulvestrant-inhibited growth: cell number (using sulforhodamine B [SRB] assay, y axis) of MCF-7 overexpressing (F) NICD4-GFP, (G) JAG1-GFP, or GFP control incubated with tamoxifen or fulvestrant for 1, 3, and 5 days (x axis) compared to the respective cell line treated with control ethanol. p values are for the 5-day treatment.

Data are represented as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01.

See also Figures S2 and S4.