Jnj 38877605

Cell Counting kit-8 (CCK-8) assays (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) were used to evaluate the proliferation of HLECs. HLECs induced by rhHGF (PeproTech, Inc., Rocky Hill, NJ, USA) or JNJ-38877605 (400 nM; Selleck Chemicals, Houston, TX, USA) were seeded in 96-well plates (2,000 cells/well, 100 µl) and incubated at 37°C for 12 or 24 h. For rhHGF, the blank control was the culture medium without cells. The negative control used the vehicle, deionized water for rhHGF and DMSO for JNJ-38877605. Cells were incubated with 10 µl CCK-8 for 2 h, and optical density (OD) was, measured at 450 nm using Varioskan Flash microplate reader (Thermo Fisher Scientific, Inc.).

The KNZ_OR cell line was established from a bone metastatic tumor at C7 that was intrinsically resistant to Osimertinib in the patient with EGFR‐mutated lung cancer and plasma EGFR‐T790M. The cells were maintained and cultured in ACL4 medium (Thermo Fisher Scientific Inc) supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (50 µg/mL) in a humidified CO₂ incubator at 37°C. H3255 and PC‐9 cells have an EGFR‐L858R point mutation and exon 19 deletion, respectively. These two cell lines were cultured in RPMI‐1640 supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (50 µg/mL) in a humidified CO₂ incubator at 37°C. Mycoplasma infection of these cells was regularly monitored using the MycoAlert Mycoplasma Detection Kit (LONZA). Cell lines were authenticated by short tandem repeat analysis at the National Institute of Biomedical Innovation in May 2015. Osimertinib, gefitinib, crizotinib, golvatinib, JNJ38877605, and foretinib were purchased from Selleck Chemicals.

To establish MET-targeted drug-insensitive cell lines, 1 × 10⁶ HepG2 cells were individually treated with a single dose of 50 nM MET-specific small molecule inhibitor JNJ-38877605 (Selleck Chemicals, S1114) and 40 μg/ml anti-MET monoclonal antibody (Alphamab, 5D5) for 3 d. After treatment, the surviving population of HepG2 cells was cultured in normal growth medium for 3 d to avoid the therapeutic stress-mediated artificial effects. The recovered HepG2 cells were treated again with JNJ-38877605 and 5D5 as above for the other 2 rounds of selection. The concentration of JNJ-38877605 and 5D5 used to select out the insensitive cell line was based on clinical reports to apply a degree of clinical relevance to this in vitro study. To assess the resistance of MET-targeted drug-insensitive cell lines, cells were plated at a concentration of 1 × 10³ cells per well in triplicate wells of 6-well tissue culture plates. Six h after plating, the triplicate wells were treated with JNJ-38877605 (0–100 nM) and 5D5 (0–80 μg/ml) for 3 d, and then subjected to cell proliferation, viability and colony formation assays as described. The resistant cells were incubated in very low concentrations of drugs to maintain their resistance in cell culture, but the drugs were withdrawn in formal experiments.