Sti571

Human recombinant Transforming Growth Factor-beta 1 (TGF-β1) and Platelet-Derived Growth Factor-BB (PDGF-BB) were obtained from Biochrom GmbH (Berlin, Germany). Fibroblast Growth Factor 2 (FGF2) and Insulin-like Growth Factor-I (IGF-I) were purchased from R&D Systems Europe (Abingdon, UK), while Epidermal Growth Factor (EGF) from Sigma (St. Louis, MO, USA). The TGF-β type I receptor (TGFβR1) kinase inhibitor SB431542 was from Sigma, while the PDGF receptor (PDGFR) kinase inhibitor STI571 from Novartis AG (Basel, Switzerland). The FGF receptor 1 (FGFR1) kinase inhibitor SU5402, the IGF-I receptor (IGFIR) kinase inhibitor tyrphostin I-OMe-AG538, and the EGF receptor (EGFR) kinase inhibitor tyrphostin AG1478 were obtained from Calbiochem-Merck KGaA (Darmstadt, Germany). The p38 MAPK inhibitor SB203580, and the NF-κB inhibitor BAY117082 were from Sigma, while the Sp1 inhibitor mithramycin from Cayman Chemical (Ann Arbor, MI, USA).

After serum starvation for 12 h, cells were incubated for 4 h with 10 ng/mL SCF
and/or 250 nM STI571 (Novartis Pharmaceuticals, Basel, Switzerland), and
immunoblot analysis was conducted as described previously51 (link).
Primary antibodies including anti-phospho-KIT, anti-total/phospho-Akt,
anti-total/phospho-S6 ribosomal protein, and β-actin antibodies
purchased from Cell Signaling Technologies (Beverly, MA), and an anti-KIT
antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

The pMX-INV, pMX-DEL^CJ, or pMX-DEL^SJ substrates were introduced in pro-B cell lines through retroviral infection, and cells that had integrated the recombination substrate were enriched based on hCD4 expression (Bredemeyer et al., 2006 (link), Liang et al., 2002 (link)). For V(D)J recombination assay, v-abl-transformed, Bcl2/pMX-INV-infected pro-B cells (10⁶/ml) were treated with 3 μM of the v-abl kinase inhibitor STI571 (referred to as ABLki in this study, Novartis) and assayed for rearrangement by FACS analysis of GFP expression or Southern blotting at 0, 72, and/or 96 hr. In some experiments, the ATM kinase inhibitor KU55933 was added at 15 μM together with STI571. 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 Biotec, 1:20 dilution). The pMX-INV-mCherry substrate was built by replacing the inverted GFP cDNA from pMX-RSS-GFP/IRES-hCD4 (pMX-INV) by an inverted mCherry cDNA.

V(D)J recombination assays were performed as previously described^{35 (link),61 (link)}. Briefly, v-Abl pro-B cells were transduced with pMX-INV retroviral vector and cells that had integrated the pMX-INV recombination substrate were enriched based on hCD4 expression using hCD4 Microbeads (Miltenyi 130-045-101). Purified pMX-INV v-Abl pro-B cells were treated with 3 μM of the Abl kinase inhibitor STI-571 (Novartis) for 72 h and assayed for recombination levels by FACS analysis of GFP/hCD4 expression (hCD4-PE antibody, Miltenyi 130-113-254, clone M-T466, 1:100). V(D)J recombination levels were scored by FACS analysis as the percentage of GFP positive cells among total hCD4 positive cells. FACS data was acquired by FACS Diva v8.0.1 (BD Biosciences) and analyzed and visualized by Flowjo v10.4.2 (TreeStar).

V(D)J recombination assays were performed as previously described^{38 (link),40} . Briefly, v-Abl pro-B cells were transduced with pMX-INV retroviral vector and cells that had integrated the recombination substrate were enriched based on hCD4 expression using hCD4 Microbeads (Miltenyi 130-045-101). Purified v-Abl infected pro-B cells (10⁶ per ml) were treated with 3 μM of the v-Abl kinase inhibitor STI571 (Novartis) for 72 h and assayed for rearrangement levels by FACS analysis of GFP/hCD4 expression (hCD4-PE antibody, Miltenyi 130-113-254, clone M-T466, 1:100). V(D)J recombination efficiency was scored as the percentage of GFP-positive cells among hCD4-positive cells. FACS data was acquired by FACS Diva v8.0.1 (BD Biosciences) and analyzed and visualized by Flowjo v10.4.2 (TreeStar). The gating strategy is provided is Supplementary Fig. 8.