Fig 2: Blocking Notch–Jag1 interactions among CLL cells does not affect IL-4-induced Notch activation, but reduces IL-4-induced Jag1 processing and cell viability.a–f CLL cells were cultured for 48 h with or without 25 ng/ml IL-4 in the presence of 60 µg/ml Jag1 neutralizing or goat IgG antibodies as isotype control (ctrl Ab) (n = 5; CLL7-8 and CLL10-12, selected to include patients with different clinical and biological characteristics). a–d Western blot analysis of Hes1 and Jag1 was performed in 25 µg whole-cell lysates, and protein loading was assessed reprobing the blots with an anti-GAPDH antibody. a, c Representative cases are shown. a Full images of Hes1 blots are shown in Supplementary Figure S7A. c For Jag1 analysis, the C-20 antibody was used. In each blot, separated panels are shown because different X-ray film exposures were necessary to detect Jag1-FL and Jag1 fragments. Full images of Jag1 blots are shown in Supplementary Figure S7B. b, d The density of the bands corresponding to Hes1, Jag1-FL, Jag1-TM, and Jag1-IC was evaluated by densitometric analysis, and densitometry units (U) were calculated relative to GAPDH. Data are the mean ± SD of five patients. ns: the difference between the two groups was not significant; *P < 0.05, **P < 0.01 according to Student’s t test. e, f Cell viability and apoptosis were evaluated by flow cytometric analysis of Annexin V/PI (An V/PI) staining. e Results are presented as the percentage of viable (An V-/PI-), early apoptotic (An V+/PI-), late apoptotic (An V+/PI+), and necrotic (An V-/PI+) cells. Representative cases are shown. f Results are presented as percentage of viable (An V-/PI-) cells and are the mean ± SD of five patients. **P < 0.01, ***P < 0.001 according to Student’s t test

Fig 3: Jag1 is constitutively processed in CLL cells.a–c Western blot analysis of Jag1 was performed in 25 µg whole-cell lysates (WCL) from primary CLL cells (n = 21), MEC1 cell line, and PBL from healthy donors (n = 3), using three different C-terminal antibodies (Abs), as indicated on top of the blots. Data in panel a are obtained using the Jag1 C-20 Ab. Data in panels b and c are from representative CLL samples where the analysis of Jag1 was repeated using the Jag1 TS1.15H (b) and Jag1 E-12 (c) Abs. WCL isolated from IM9 cell line was used as a positive control for Jag1 expression. Protein loading was assessed reprobing the blots with an anti-GAPDH antibody. In each blot, separated panels are shown because different X-ray film exposures were necessary to detect Jag1-FL and Jag1 fragments. Positions of the molecular weight markers (kDa) are indicated on the right of the blots. In the blots with PBL, an empty lane has been placed between PBL2 and CLL11 to avoid cross-contamination. Vertical line inserted in the middle blot of panel b indicates a repositioned gel lane. Full images of all blots are shown in Supplementary Figure S1

Fig 4: Effects of IL-4 on Jag1 expression in CLL cells are mediated by the PI3Kd/AKT signaling.a Western blot analysis of AKT Ser473 phosphorylation (phospho-AKT) was performed in 25 µg whole-cell lysates from CLL cells cultured with or without 25 ng/ml IL-4 for the indicated times. Protein loading was assessed using anti-total AKT and anti-GAPDH antibodies. One representative case is shown. Full image of the blot is shown in Supplementary Figure S5A. b–e CLL cells, pretreated for 2 h with the PI3Kd inhibitor Idelalisib (5 µM) or 0.01% DMSO as control, were incubated for further 24 h with or without 25 ng/ml IL-4 (n = 6; CLL10 and CLL12-16, selected to include patients with different clinical and biological characteristics). b Western blot analysis of Jag1 was performed, using the C-terminal Jag1 C-20 antibody, in 25 µg whole-cell lysates, and protein loading was assessed reprobing the blots with an anti-GAPDH antibody. Representative cases are shown. Full images of the blots are shown in Supplementary Figure S5B. c Blots of each sample were subjected to densitometric analysis, and densitometry units (U) were calculated relative to GAPDH. Data are the mean ± SD of six patients. ns: the difference between the two groups was not significant; **P < 0.01 according to Student’s t test. d, e Cell viability and apoptosis were evaluated by flow cytometric analysis of Annexin V/PI (An V/PI) staining. d Results are presented as the percentage of viable (An V-/PI-), early apoptotic (An V+/PI-), late apoptotic (An V+/PI+), and necrotic (An V-/PI+) cells. Representative cases are shown. e Results are presented as percentage of viable (An V-/PI-) cells and are the mean ± SD of six patients. *P < 0.05, ***P < 0.001 according to Student’s t test

Fig 5: IL-4 enhances Jag1 expression in CLL cells.a–f CLL cells were cultured for 24 h with or without 25 ng/ml IL-4 (n = 6; CLL7-12, selected to include patients with different clinical and biological characteristics). a, b Western blot analysis of Jag1 was performed, using the C-terminal Jag1 C-20 antibody, in 25 µg whole-cell lysates, and protein loading was assessed reprobing the blots with an anti-GAPDH antibody. a In each blot, separated panels are shown because different X-ray film exposures were necessary to detect Jag1-FL and Jag1 fragments. Representative cases are shown. Full images of the blots are shown in Supplementary Figure S4A. b The density of the bands corresponding to Jag1-FL, Jag1-TM, and Jag1-IC was evaluated by densitometric analysis, and densitometry units (U) were calculated relative to GAPDH. Data are the mean ± SD of six patients. **P < 0.01, ***P < 0.001 according to Student’s t test. c, d Western blot of Jag1-IC was performed, using the C-terminal Jag1 C-20 antibody, in 25 µg nuclear extracts. Protein loading and adequate fractionation were assessed using anti-lamin B1 and anti-ß-tubulin antibodies. c Representative cases are shown. Full images of the cropped blots are shown in Supplementary Figure S4B. d Blots of each sample were subjected to densitometric analysis, and densitometry units (U) were calculated relative to lamin B1. Data are the mean ± SD of six patients. ***P < 0.001 according to Student’s t test. e mRNA levels of Jag1 were evaluated by quantitative real-time PCR, normalized to GAPDH and represented as fold change with respect to untreated cells. Results are the mean ± SD of six patients. *P < 0.05 according to Student’s t test. f Cell viability and apoptosis were evaluated by flow cytometric analysis of Annexin V/PI (An V/PI) staining. Results are presented as percentage of viable (An V-/PI-) cells and are the mean ± SD of six patients. ***P < 0.001 according to Student’s t test