Fig. 2
The loss of NuRD interaction with mutant NOTCH1 over-activates a subset of genes critical for the survival of CLL cells. a Heatmap showing the nine upregulated genes related to the cytokine signaling pathway in ICN-delCT variants. Color indicates normalized expression level (log); Red corresponds to higher expression, and green corresponds to lower expression. b Ccl17 and Ccl22 mRNA levels in Baf3 cells transfected with the indicated plasmids. Values were normalized against Gapdh. c The transcriptional level of CCL17 and CCL22 genes in human CLL patients with WT NOTCH1 (CLL-WT, n = 18) or NOTCH1-delCT mutation (CLL-delCT, n = 7). d Primary CLL cells were infected with the indicated lentiviral vectors. Immunoblots of the indicated proteins were detected. e CCL17 and CCL22 mRNA levels in primary CLL cells described in d. Values were normalized against GAPDH. f Schematic of the CCL17 promoter indicating the CSL-binding sites (upper panel). ChIP assays detected NOTCH1-ICN, MTA2, and HDAC1 on CCL17 gene promoter performed in primary cells of the indicated CLL patients (lower panel). ChIP results are presented as the mean of experiments performed in three independent samples. g The chemokine CCL17 concentration in the serum of human CLL patients with WT NOTCH1 or NOTCH1-delCT was detected by ELISA. h The chemotaxis assay for cellular supernatants derived from Baf3 cells expressing ICN or ICN-delCT. Transwell chambers were used to detect T-cell migration ability and the number of migrated cells was counted manually. i The chemotaxis assay for the serum of human CLL patients with WT (n = 3) or NOTCH1-delCT (n = 3). j A schematic model illustrating the induction of CCL17 by NOTCH1-delCT mutation in CLL. CoA, co-activator. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant; two-tailed t-test. Data represent means of triplicate reactions ± sem
