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. Author manuscript; available in PMC: 2020 Aug 1.
Published in final edited form as: Mol Cancer Res. 2019 May 14;17(8):1639–1651. doi: 10.1158/1541-7786.MCR-18-1292

Figure 3. R72 has greater transcriptional activity toward Notch1.

Figure 3.

(A) q(RT)-PCR of (i) NOTCH1 and HES1 relative to GAPDH in R72 and P72 NHF cells; (ii) Expression of Notch1 and Hes1 in P72 and R72 NHF cells by Western immunoblotting.

(B-C) Expression of NOTCH1 and HES1/5 in P72 and R72; (B) H1299 cells in the presence of doxycycline (dotted line-expression of Notch1 in non-treated H1299 cells) or in (C) CRL4004 angiomyolipoma-derived cells by (B-i, C-i) q(RT)-PCR relative to GAPDH and (B-ii, C-ii) Western immunoblotting.

(D-E) Expression of TP53, NOTCH1 and HES1 in (D) 621-101 and (E) CRL4004 cells depleted of p53 using shRNA by (D-i, E-i) q(RT)-PCR relative to GAPDH and (D-ii, E-ii) Western immunoblotting.

(F) Expression of Notch1 and Hes1 in kidneys of P72 and R72 mice by (i) q(RT)-PCR relative to GAPDH, or (ii) Western immunoblotting, (iii) Quantification of data in F-ii by densitometry.

(G) Binding of P72 and R72 to p53 responsive element (p53-RE, P53A) within the Notch1 promoter in (i) NHF, (ii) doxycycline treated H1299, (iii-iv) 621101 and CRL4004 cells and (v) in kidneys of P72 and R72 mice by ChIP-qPCR. Binding is shown as fold enrichment over the IgG (dotted line).

Data represent means±SEM. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.

Data are representative of four, n=4 (A-i), , n=14 (Hupki P n=7 mice; Hupki R n=7 mice) (F-ii), n=5 (Hupki P n=2 mice; Hupki R n=3 mice) (F-i), n=5 (G-v), five (A-ii), three, n=3 (B, C, D, E), and two (G-i, n=4 and G-ii-G-iv n=3) independent experiments.