Del 22379

To induce RTK signaling, HEK293T cells were treated with 0.1 μg/ml of recombinant human EGF (AF-100-15A; Peprotech, Cranbury, NJ, United States) dissolved in phosphate-buffered saline (PBS) containing 0.2% bovine serum albumin. The conditions for the kinase inhibitor treatment were determined based on previous studies: SCH772984 (S7101, SelleckChem, Houston, TX, United States): 10 μM for 2 h (Martinez et al., 2021 (link)), DEL22379 (S7921, SelleckChem): 10 μM for 30 min (Herrero et al., 2015 (link)), LJH685 (S7870, SelleckChem): 10 μM for 3 h (Ren et al., 2020 (link)), and dasatinib (73082, STEMCELL Technologies, Vancouver, Canada): 200 nM for 1 h (Koreckij et al., 2009 (link)). To inhibit the proteasome complex, the cells were treated with 10 μM MG132 (C2211, Sigma–Aldrich, St. Louis, MO, United States) for 6 h.

The ERK1/2 inhibitor LTT462 and ruxolitinib were provided by Novartis via Material Transfer Agreement, MK-8353, SCH772984 and DEL-22379 purchased from Sellekchem. LTT462 was administered by gavage at 75 mg/kg qd, ruxolitinib at 60 mg/kg bid and MK-8353 at 30 or 40 mg/kg bid. Inhibitors for in vitro use were stored at −20 °C in DMSO.

MDAMB231 cells were purchased from ATCC and grown in DMEM (Gibco) media supplemented with 10% FBS, 1% Pen/strep, and 1% Glutamax Supplement (Thermo Fisher) and maintained at 37 °C in 5% CO₂. SUM159 cells were purchased from Asterand Bioscience and grown according to manufacturer’s suggestion. Recombinant human EGF was used at 10 nM. Batimastat (BB94, Tocris Bioscience) was used at 10 μM. Selumetinib (AZD6244, Selleck Chem) was used at 1.5 μM. Binimetinib (Mek162, ARRY-162, Selleck Chem) was used at 1.2 μM. PD0325901 (Selleck Chem) was used at 33 nM. Ulixertinib (BVD-523, VRT752271, Selleck Chem) was used at 30 nM. DEL-22379 (Selleck Chem) was used at 5 μM. GDC-0994 (Selleck Chem) was used at 30 nM. JQ1 (Tocris Bioscience) was used at 0.2 μM. R428 (BGB324, Selleck Chem) was used at 1 μM. DMSO at matched concentrations was used as all controls.