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. 2017 Nov 29;8:1848. doi: 10.1038/s41467-017-01845-1

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commonslicense, unless indicated otherwise in a credit line to the material. If material is not included in the article’sCreative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — Notch1 directly activates the Rheb promoter. a q(RT)-PCR of (i, iii) RHEB and (ii, iv) HES1 relative to GAPDH in synchronized (i, ii) 621–101 and (iii, iv) CRL4004 angiomyolipoma cells in DMEM. The statistical significance of RHEB and HES1 expression at different time points was determined relative to time 0. b Expression of Rheb and Hes1 in synchronized (i) 621–101 and (ii) CRL4004 angiomyolipoma cells in DMEM by western immunoblotting. Numeric values represent densitometry analysis of the expression of Rheb relative to expression of Gapdh. c (i) Rheb promoter: triangles indicate potential RBPJ binding sites with a reverse (1, 2) and a forward (3, 4) orientation. (ii–v) Binding of Notch1 to (ii, iv) the potential RBPJ binding site 1, NRE2, NRE3, and the potential RBPJ binding site 4 of RHEB or (iii, v) HES1 promoter in non-synchronized (ii, iii) 621–101 or (iv, v) CRL4004 cells grown in DMEM by ChIP-qPCR; binding is shown as fold enrichment over the IgG (dotted line). d (i) Wild-type Rheb-luciferase (RHEB), RHEB-RBPJ1- (RBPJ1), RHEB-NRE2- (NRE2), RHEB-NRE3- (NRE3), or RHEB- RBPJ4- (RBPJ4) mutant-luciferase or control-luciferase (control) promoters activity in HeLa cells. (ii) Expression of N1ICD, MAML1, Hes1, Rheb, and phospho-S6K in 293T, HeLa, CRL4004, and H1299 cells. (iii) Expression of cleaved Notch1 (Val1744), Notch1, Hes1, Rheb in 621–101, CRL4004, and H1299 cells depleted of Notch1 using shRNA by western blotting or (iv) q(RT)-PCR. Numeric values represent densitometry analysis of the expression of Rheb relative to expression of Gapdh. (iv) NOTCH1, RHEB, and HES1 relative to GAPDH in 621–101 cells depleted of Notch1 using shRNA. e Binding of Notch1 to the (i) potential RBPJ1 site, (ii) NRE2, and (iii) NRE3 within Rheb or (iv) Hes1 promoter in synchronized 621–101 cells in DMEM by ChIP-qPCR as in (c (ii, iii)). Data represent means ± s.e.m. Error bars are defined as means + s.e.m. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, t-test. Data are representative of three (a (iii, iv), b, d (i–iv)), four (e), and six (a (i, ii), c (ii, iii)) independent experiments