Fig. 2

Intracellular NOTCH1 directly binds to and activates SHQ1. a Seven T-ALL cell lines and four primary T-ALL cells were subjected to GSI (Compound E, 1 μM) or DMSO treatment for 24 h. SHQ1 mRNA and protein levels were subsequently determined by quantitative polymerase chain reaction (qPCR) and immunoblots. b Correlation of SHQ1 expression with NOTCH1 in 174 primary T-ALL samples (GSE13159) with mRNA levels presented as log2 median-entered intensity. Pearson’s correlation coefficient (R) = 0.481, p < 0.001, paired t-test. c Immunoblots of SHQ1 and ICN1 in additional 11 primary T-ALL and 3 normal thymus samples. d Chromatin landscapes around the SHQ1 locus in HPB-ALL (GSE58406) and CUTLL1 (GSE51800) cells. The associations of nuclear NOTCH1 and RBPJ with the SHQ1 transcriptional start region are shown with respect to NOTCH1 active or inactive state. e Binding of ICN1 to the SHQ1 or HES1 promoter was analyzed by ChIP in SIL-ALL cells with or without GSI treatment (Compound E, 1 μM) for 24 h. Averages of fold enrichment between ICN1 and isotype IgG are shown. α-N1 denotes antibodies against ICN1. f Schematic presentation of two potential RBPJ-binding sites proximal to the SHQ1 transcription initiation site. Luciferase assays were carried out with SHQ1 DNA sequence (−441 to −194) or HES1 promoter cloned into pGL3-basic vector; reporter activities relative to empty pGL3 vector without ICN1 are presented as average fold induction. Data shown represent the means ( ± SEM) of three biological replicates; *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t-test (a, e, f)