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

Fig. 9. Signaling pathways involved in Notch1 and Notch2 upregulation induced by IL-4 in CLL cells.

Fig. 9

CLL cells (n = 5; CLL7-8 and CLL10-12, selected to include patients with different clinical and biological characteristics) were preincubated for 2 h with 5 µM Idelalisib or 0.01% DMSO (a, b), or with 10 µM Rottlerin or 0.05% DMSO (c–f), and then were cultured with or without 25 ng/ml IL-4 for further 24 h. a–d Western blot analysis of Notch1 and Notch2 was performed in 25 µg whole-cell lysates, and protein loading was assessed using an anti-GAPDH antibody. a, c Representative cases are shown. Full images of the blots are shown in Supplementary Figure S9A–C. b, d The density of the bands corresponding to Notch1-TM, Notch1-IC, Notch2-TM, and Notch2-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, ***P < 0.001 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 < 0.05, **P < 0.01, ***P < 0.001 according to Student’s t test