Fig. 7. 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