Extended Data Figure 9: Ibrutinib increases AID expression and the frequency of translocations to AID on- and off-target sites in mouse activated B cells.
From: Phosphatidylinositol 3-kinase δ blockade increases genomic instability in B cells
a, AKT phosphorylation was detected by western blot in mouse activated B cells treated with DMSO, idelalisib, duvelisib or ibrutinib (1 μM) for the indicated time points (n = 2 biological replicates). For gel source data, see Supplementary Fig. 1. b, MEC1 and Mino human lymphoma cells were treated with the indicated inhibitors (1 μM) and AKT phosphorylation was evaluated by western blot (n = 3 biological replicates). c, Viable cells were counted at the indicated time points by Trypan Blue exclusion in activated B cells treated with DMSO or ibrutinib (1 μM). Data are expressed as mean ± s.d. (n = 3). P values calculated by two-tailed Student’s t-test. d, Western blot for AID protein expression in activated B cells treated with 1 μM ibrutinib. The DMSO panel from Fig. 1a is shown for comparison (n = 3 biological replicates). e, Aicda mRNA levels analysed by qRT–PCR in activated B cells treated with DMSO or ibrutinib (1 μM). f, IgG1 CSR in activated B cells analysed by flow cytometry. Data are expressed as mean ± s.d. (n = 3 biological replicates). *P < 0.05, ***P ≤ 0.001, two-tailed Student’s t-test (e, f). g, Histograms showing translocation junction frequency to AID on-target (#, Igh and Igk loci) and off-target sites in activated B cells treated with ibrutinib. Targets are divided on the basis of the statistical significance of increased junctions frequency compared to DMSO treatment (FDR < 0.001 on the left; FDR < 0.01 on the right). Statistical analysis is indicated in the Method. For each treatment, data are from pooled HTGTS libraries of similar size from independent experiments as indicated in Supplementary Tables 1, 5. h, Venn diagrams showing the fraction of AID off-target sites shared in activated mouse B cells treated with ibrutinib, idelalisib or duvelisib.
