Ljh685

HEK293 cells and HeLa cells were obtained from the American Type Culture Collection without further authentication. eHAP1 human cell line was purchased from Horizon Discovery. Human cells were maintained in 5% CO₂ at 37°C. HEK293 and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and eHAP1 cells in Iscove Modified Dulbecco Media (IMDM). Both media were supplemented with 10% Fetal Bovine Serum, 1% Penicillin-Streptomycin, 1% Pyruvate. When indicated, cells were treated with Phorbol 12-Myristate 13-Acétate (Fisher Scientific, Cat#10061403), Human EGF (Euromedex, Cat# HC88823), LJH685 (Selleck Chemicals, S7870), BI-D1870 (Selleck Chemicals, S2843) and/or PD184352 (Selleck Chemicals, S1020). For transient plasmid expression, cells were transfected using either Jet Prime reagent or calcium phosphate precipitation. For shRNA-mediated RSK1/2 knockdown, cells were infected by lentiviruses produced with vectors from the Mission TRC shRNA library (RSK1, TRCN470; RSK2, TRCN537) in the presence of 4 mg/ml polybrene and selected 48 h after infection with 2 μg/mL puromycin.

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.

ON-TARGETplus SMARTpool small interfering RNAs (siRNAs) were purchased from Dharmacon: ID1 (#L-005051-00-0005), ID3 (#L-009905-00-0005), ROCK1 (#L-003536-00-0005), p120 (#L-012572-00-0005), YAP (#L-012200-00-0005), non-targeting control (#D-001810-10-50). Cells were reverse-transfected with 1 pmol/10 nM of each siRNA in 96-well plates using Lipofectamine RNAiMAX (Life Technologies) and Opti-MEM (Thermo Fisher Scientific) following manufacturer’s instructions. pCDNA3 backbone was from Invitrogen and hE-cadherin-pcDNA3 was a gift from Barry Gumbiner (Addgene plasmid # 45769). Plasmids were forward transfected into cells using FuGENE 6 (Promega). BEZ-235, LY-294002, GSK-1120212, SCH-772984 and LJH-685 were purchased from Selleck Chemicals. Y-27632 was purchased from Sigma Aldrich.