(A) NF-κB luciferase reporter activity was detected in mice transplanted with: ICN1-GFP (WT), ICN1-GFP (NFκB-ER-luc) or GFP (NFκB-ER-luc). Bioluminescent imaging of mice 9 and 21 days after transplantation is shown. (B) Peripheral blood CD4/CD8 FACS analysis; (C) qPCR from sorted cells as indicated. Error bars denote +/− Standard Deviation. (D) Protein levels of Notch1-IC (ICN1) and Lamin A/C in nuclear extracts of thymocytes from Mx-Cre (Mx) and Ef1α-ICN1, Mx-Cre (ICN1) mice 4 weeks after pIpC injection. (E) NF-κB DNA binding activity was analyzed by EMSA using nuclear extracts of thymocytes from MxCre, Ef1α-ICN1 Mx-Cre mice 4 weeks after pIpC injection and wt thymocytes either untreated (Co) or treated with mTNFα. (F) Heat-map shows expression of NF-κB target genes selected from the literature (http://www.nf-kb.org) up-regulated in leukemic DP cells compared to control thymic DP cells by unpaired one-side t-test with α=0.05. See also related figure S1.

(A) Luciferase assay with the 3xκB-luc-reporter in HEK-293T cells transfected with a doxycycline-inducible Hes1 construct. Induction of Hes1 after doxycycline treatment is shown in the lower panel. (B) Immunofluorescence of HEK-293T transfected with flag-Hes1 showing the expression pattern of transfected Hes1 and endogenous p65. (C) Western blot of endogenous IκBα in HEK-293T cells transfected with inducible Hes1 in the presence or absence of TNFα. (D) Lack of IκBα immunostaining in cells transfected with ectopic Hes1. (E) Quantitative RT-PCR to determine the expression levels of IκBα mRNA in the presence or absence of Hes1. Error bars denote +/− Standard Deviation. (F) Western blot of IκBα levels in HEK-293T transfected with the doxycycline-inducible Hes1 construct in the presence or absence of the proteasome inhibitor MG132. (G) Western blot showing the levels of IκBαwt or IκBα_K21-22R cotransfected with inducible Hes1. See also related figure S2.

Western blots of cells transfected with an inducible Hes1 vector and treated for 16h with doxycycline and TNFα as indicated. (A) HEK-293T cells were treated with the IKK inhibitor BAY65-1185, (B) Cells were cotransfected with HA-IKKα or HA-IKKβ, (C) IKKβ-deficient cells were reconstituted with HA-IKKβ and (D) HEK-293T cells were transfected with the dominant negative TAK1 (K63W) construct. See also related figure S3.

(A) Jurkat cells transduced with scrambled shRNA or Hes1-shRNA (80 to 90% reduction in Hes1 protein levels) were analyzed for the levels of IκBα protein during TNFα treatment, (B) IκBα is restored in the presence of GSI (L685,458) (C) levels of P-IKK in control and Hes1 knockdown cells by western blot and (D) expression of NF-κB-target genes by qRT-PCR in Jurkat or (E) primary human T-ALL leukemic cells transduced with the control or Hes1-shRNA lentivirus. (F) Experimental design to test the contribution of Hes1 to ICN1 transduced cells. (G–I) Primary mouse Lin⁻ bone marrow progenitors transduced with the indicated lentivirus. (G) Colonies obtained from serial replatings on methylcellulose were counted. (H) T-cell differentiation was determined by CD3 expression after 7 days in co-culture on OP9-Dll1. One representative experiments out of three is shown. (I) Expression levels of the indicated genes were tested by qRT-PCR after 2 weeks in culture. (J) Number of colonies obtained in serial plating from ICN1 transduced cells in the presence or absence of BAY65-5811. Error bars denote +/− Standard Deviation. See also related figure S4.

(A) Levels of A20, CYLD and Hes1 mRNA in primary samples for normal WPB (1–9), purified normal CD3+ cells from PB (10–13) and PB from T-ALL patients (1–33) by qRT-PCR. (B) Analysis by qRT-PCR of CYLD or A20 levels in cells transduced with scrambled or Hes1-shRNA. For both A–B panels, error bars denote +/− Standard Deviation. (C–D) Expression of CYLD and A20 measured by qRT-PCR (C) or western blot (D) in HEK-293T cells expressing ectopic Hes1 untreated or treated with TNFα. (E) Western blot analysis of CYLD, active Notch1 and Hes1 protein levels in different leukemic cell lines. See also related figure S5.

(A) Scheme of the CYLD promoter indicating the putative Hes1 binding sites. (B) Detection of the CYLD PRO-2 Hes1 binding site by PCR using the indicated antibodies. (C) Relative enrichment of the CYLD promoter quantified by qPCR from ChIP with anti-Hes1 antibody in Jurkat T-ALL cells transduced with scrambled or Hes1-shRNA. Error bars denote +/− Standard Deviation. (D) Primary mouse Lineage-negative bone marrow progenitors were transduced with the indicated lentivirus and colonies obtained from serial replating on methylcellulose were counted every week. (E) Representative FACS analysis from week 3 progenitors after 7 days on OP9-Dll1 stroma. (F) EPITYPER methylation analysis showing no significant hyper-methylation of the CYLD promoter. Patient samples were analyzed utilizing the EpiTYPER Sequenom methylation assay. The heat map illustrates the range of methylation percentages, with red indicating 0% methylation and yellow indicating 100% methylation. Numbers along the top indicate CpG dinucleotide, patient sample numbers are listed along the vertical axis (p denotes the identity of the patient as presented in Figure 5A). Samples are run in duplicate. Two primers were run for each region. The positive control, IVD, has near 100% methylation, while the negative control, WGA, is unmethylated. See also related figure S6.

Human T-ALL cell lines were treated with 100 µM of control (Co) or NBD inhibitory peptide for 24 hours. (A) Nuclear extract from treated and untreated cells were isolated and checked for NF-κB-DNA binding activity by EMSA. Untreated cells (Ø) are also shown. (B) Bar-graph indicating the proportion of Annexin V positive cells 24h after the NBD treatment. Error bars denote +/− Standard Deviation. (C) Caspase-3 activation as detected by FACS with fluorogenic cell permeable active caspase 3- specific probe (Fam-DEVD-fmk) in 3 representatives T-ALL lines 24h post treatment. All experiments shown in A–C were repeated at least three times. (mean ± SD of measurements: *, p<0.05; **, p<0.01 Student’s t test analysis). (D) Heat map showing up changes in expression of selected genes in response to NBD peptide treatment. See also related figure S7.

(A–B) Peripheral blood FACS analysis using CD4, CD8 and Ly5.2 antibodies at different time points post transplantation. (C) ΔEN1-luc Nemo^wt/wt, Mx-Cre and ΔEN1-luc Nemo^f/f, Mx-Cre chimeras were monitored before and after pI-pC injections using in vivo imaging system (IVIS) to quantify luciferase intensity. 2 weeks post-transplantation and before any deletion of NEMO, both chimeras harbored homogenous tumor load. Once this was verified mice were injected by pI-pC. (D) Quantification of luciferase intensity (dorsal and ventral) at different time-points. Error bars denote +/− Standard Deviation. (E) Determination of apoptotic cells gated on GFP⁺ cells 5 days after pIpC injections in ΔEN1-GFP Nemo^wt/wt, Mx-Cre and ΔEN1-GFP Nemo^f/f, Mx-Cre chimeras. (F) Micrographs of hematoxylin/eosin-stained liver and spleen sections prepared 40 days after transplantation. (G) Kaplan-Meyer survival curve of the different recipient animals (p<0.005). See also related figure S8.