Pd180970

The pMX-INV or pMX-DEL^CJ substrate was introduced in pro-B cell lines through retroviral infection and cells that had integrated the recombination substrate were enriched based on hCD4 expression7 (link)34 (link). For V(D)J recombination assay, v-abl transformed Bcl2/pMX-INV infected pro-B cells (10⁶ per ml) were treated with 3 μM of the v-abl kinase inhibitor STI571 (Novartis) or 0.3 μM of the STI571-analogous v-abl kinase inhibitor PD180970 (Sigma) and assayed for rearrangement by FACS analysis of GFP expression or Southern blotting at 0, 72 or 96 h. In some experiments, the ATM kinase inhibitor KU55933 was added at 15 μM together with PD180970 or STI571 (named ABLki). For FACS analysis, V(D)J recombination efficiency was scored as the percentage of GFP positive cells among hCD4-positive cells (human CD4-PE, Miltenyi, 1:20 dilution). For ABLki release experiments, cells were collected, washed and cultured without ABLki for 3–4 days before metaphases preparation.

The following compounds were used: oryzalin (36182; Sigma-Aldrich), 5-iodotubercidin (5-ITu) (I100; Sigma-Aldrich), PD-180970 (PZ0142; Sigma-Aldrich), PD-166326 (9000988; Cayman Chemical), PD-173955-Analog1 (SYN-1062; SYNkinase), ponatinib (CS-0204; CHEMSCENE), bosutinib (PZ0192; Sigma-Aldrich), bafetinib (A10119; AdooQ Bioscience), PP2 (P0042; Sigma-Aldrich), PP1 (BML-EI275; Enzo Life Sciences), 4-aminopyrazolo[3,4-d]pyrimidine (PP2-Analog1) (A1041; Tokyo Chemical Industry), 1-tert-butyl-1H-pyrazolo[3,4-d]pyrimidine-4-amine (PP2-Analog2) (GF-0723; Key Organics), PP3 (A2737; Tokyo Chemical Industry), and Src inhibitor1 (sc-204303; Santa Cruz Biotechnology) (Karni et al, 2002 (link); Wisniewski et al, 2002 (link); Hum et al, 2014 (link); Rossari et al, 2018 (link)).
Each compound was dissolved in DMSO and used at a final concentration of 10 μM in 0.1% DMSO, except for the dosage assay, and in oryzalin, which was used at 1 μM. oryzalin (10 μM) was used in the P. patens assay.