The expression of PI3Kγ (p110γ) on the MCL cell lines and PBMC from MCL patients was analysed by western blotting. Primary cells were purified by MACS (Miltinyi Biotech, Woking) to remove non-B cells as T cells and monocytes are known to express PI3Kγ (> 99% purity; data not shown). The monocyte cell line THP-1 was used as a positive control for PI3Kγ expression.
In order to determine the efficacy of the PI3K inhibitors MCL cells were incubated for 1 h with inhibitors to different PI3K isoforms [PI3Kα (A66), PI3Kδ (idelalisib), PI3Kγ (CZC24832) (all from Selleck, Cambridge UK), and the dual PI3Kδ/γ inhibitor [(duvelisib) Verastem Oncology]. The effects of the inhibitors on the phosphorylation of AKT in response to BCR cross-linking with goat anti-human IgM [F(ab)2 fragments; 20 μg/ml; Jackson Immunoresearch, Pensylvania)] and chemokine signalling with CLL21 [(1 µM) Biotechne, Abbingdon] were then examined by western blotting. Inhibitors were titrated using the following concentrations 100 nM; 500 nM; 1 μM and 2 μM. AKT phosphorylation was inhibited by 1 μM (Supplementary Figure1 F) which is below the peak plasma concentration for idelalisib and duvelisib33 (link),34 (link).
In order to determine the efficacy of the PI3K inhibitors MCL cells were incubated for 1 h with inhibitors to different PI3K isoforms [PI3Kα (A66), PI3Kδ (idelalisib), PI3Kγ (CZC24832) (all from Selleck, Cambridge UK), and the dual PI3Kδ/γ inhibitor [(duvelisib) Verastem Oncology]. The effects of the inhibitors on the phosphorylation of AKT in response to BCR cross-linking with goat anti-human IgM [F(ab)2 fragments; 20 μg/ml; Jackson Immunoresearch, Pensylvania)] and chemokine signalling with CLL21 [(1 µM) Biotechne, Abbingdon] were then examined by western blotting. Inhibitors were titrated using the following concentrations 100 nM; 500 nM; 1 μM and 2 μM. AKT phosphorylation was inhibited by 1 μM (Supplementary Figure
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