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. 2015 Jan 2;125(2):621–635. doi: 10.1172/JCI78782

Figure 6. NOTCH accumulation in FBXW7-deficient BMSCs promotes CCL2 expression and cancer metastasis.

Figure 6

(A) Immunoblot analysis of FBXW7 substrates in the indicated BMSCs. (B) Relative abundance of Ccl2 mRNA in WT BMSCs infected with retroviruses encoding NICD1, c-MYC, or KLF5. (C) Luciferase assay for the Ccl2 gene in BMSCs infected with retroviruses for NICD1, c-MYC, or KLF5. (D) Relative abundance of Ccl2 mRNA in CAG-Cre-ERT2 Fbxw7Δ/Δ BMSCs incubated with DAPT. (E) WT and mutant forms of the mouse Ccl2 gene promoter fused to the firefly luciferase gene. Consensus binding sequences for NOTCH–RBP-Jκ are shown in bold. Proximal and distal amplicons in G are indicated. (F) Luciferase assay for the Ccl2 gene in CAG-Cre-ERT2 Fbxw7Δ/Δ BMSCs. (G) ChIP analysis of the Ccl2 gene promoter. Immunoprecipitation was performed with antibodies against NOTCH1 or with control IgG. (H and I) Intravenous transplantation with B16F10 cells for Fbxw7fl/fl (n = 8), Fbxw7fl/fl RbpJκfl/fl (n = 11), Mx1-Cre Fbxw7Δ/Δ (n = 5), and Mx1-Cre Fbxw7Δ/Δ RbpJκΔ/Δ (n = 8) mice. (J and K) Intravenous transplantation with B16F10 cells for Fbxw7fl/fl (n = 8), Fbxw7fl/fl c-Mycfl/fl (n = 7), Mx1-Cre Fbxw7Δ/Δ (n = 6), and Mx1-Cre Fbxw7Δ/Δ c-MycΔ/Δ (n = 8) mice. Gross appearance of the lungs (H and J) and their occupancy by B16F10 colonies (I and K) are shown. (L) Serum concentration of CCL2, determined by ELISA. Scale bars: 10 mm (H and J). Data are mean ± SD (n = 3) (B–D, F, G, and L); horizontal bars in I and K indicate means. **P < 0.01, ***P < 0.001, 1-way ANOVA and Bonferroni test (B–D, I, K, and L).