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. 2018 Nov 26;9(12):1160. doi: 10.1038/s41419-018-1185-6

Fig. 5. Effects of IL-4 on Jag1 expression in CLL cells are mediated by the PI3Kδ/AKT signaling.

Fig. 5

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 PI3Kδ 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