Bosutinib

Cochlear explants from E15.5 embryos were established as previously described (Lee et al., 2012 (link)). Explants were treated with either vehicle (DMSO) or 5 μM SU6656 (Millipore) after four hours in vitro. After 48 hours of drug treatment, media were replaced with drug-free media. Explants were maintained for another three days in vitro and then processed for immunostaining. For in vivo Src inhibition, a single dose of Bosutinib (LC laboratories) was administered intraperitoneally to wild-type dams on E16.5 or 17.5 at 30 mg/kg, and embryos were harvested four hours later and processed for immunostaining. Embryos from vehicle-injected dams were used as controls.
Western blot analysis of cochlear tissues was performed as previously described (Lee et al., 2012 (link)). Briefly, lysates from two cochleae were pooled and loaded in each lane. GAPDH served as loading control. To assay for Src inhibition, cochlear explants were established on E16.5, treated with DMSO, SU6656 or Bosutinib at 5 μM for 1 hour, lysed in SDS sample buffer and analyzed by pY416-Src immunoblotting.

3-Deazaneplanocin A (DZNEP), a pharmacological inhibitor of enhancer of zeste homolog 2 (EZH2), the catalytic subunit of PRC2, was purchased from Cayman Chemical (Cat #13828, Ann Arbor, MI) [14 (link)]. Bosutinib, an inhibitor of SRC kinase, was purchased from LC Laboratories (Cat #B1788, Woburn MA) [15 (link)]. SB203580, a p38-specific inhibitor was purchased from InvivoGen (Cat #tlrl-sb20, San Diego CA) [16 (link)]. Recombinant human TNF-α was purchased from R&D Systems (Cat #210-TA-005, Minneapolis MN). Paclitaxel, a therapeutic inhibitor of mitosis, was purchased from Sigma (Cat #T1912, St. Louis MO).

All common reagents such as dimethyl sulfoxide (DMSO) were reagent-grade quality and obtained from Thermo Fisher Scientific (Waltham, MA, USA) or Sigma-Aldrich (St. Louis, MO, USA). Tissue culture media, fetal bovine serum (FBS) and supplements were purchased from Thermo Fisher Scientific. The CellTiter-Glo™ viability assay kit (cat# G7573) was obtained from Promega (Madison, WI, USA). Dasatinib and bosutinib were obtained from LC Laboratories (Woburn, MA, USA) and Selleck Chemicals (Houston, TX, USA), respectively. The manufacturer asserted that this was genuine bosutinib and, in addition, we subjected our lot of bosutinib to NMR analysis and confirmed the structure of the bosutinib used in this report as being genuine bosutinib and not an isomer (34 (link)).

Bosutinib, masitinib and midostaurin (PKC412) were purchased from LC Laboratories (Woburn, MA, USA), ponatinib from Selleck Chemicals (Houston, TX, USA), piceatannol and pimozide from Sigma Aldrich (San Louis, MO, USA), and dasatinib, sorafenib, sunitinib, tozasertib, vorinostat, everolimus (RAD001), erlotinib, gefitinib, and lapatinib from ChemieTek (Indianapolis, IN, USA). Imatinib, nilotinib, and BEZ235 were kindly provided by Dr.E.Buchdunger and Dr.P.W.Manley (Novartis, Basel, Switzerland). Stock solutions of drugs were prepared by dissolving in DMSO (Merck, Darmstadt, Germany). A specification of targeted drugs is shown in Supplementary Table S1. RPMI 1640 medium and fetal calf serum (FCS) were from PAA Laboratories (Pasching, Austria), ³H-thymidine from Amersham (Buckinghamshire, United Kingdom), and the Annexin V-FITC Kit from eBiosciences (San Diego, CA, USA). Recombinant human (rh) stroma cell-derived factor-1 alpha (SDF-1α) and rh eotaxin were purchased from R&D Systems (Minneapolis, MN, USA), and rh interleukin-5 (IL-5) from BD Bioscience (San José, CA, USA). A specification of monoclonal antibodies (mAb) used in this study is shown in Supplementary Table S2.