Unc0638

E67 was synthesized in accordance to the methods described by Chang et al (22 (link)). UNC 0638 was purchased from the Cayman Chemical Company (Ann Arbor, MI) (23 (link)). Both drugs were dissolved in DMSO and stored at −20°C. DAOY, DAOY-REST cells (3×10⁴), UW426 (4×10⁴) and D283 (7.5×10⁴) were seeded at the indicated densities in 96-well plates containing 100 μl of media. After overnight incubation, cells were treated for 24, 48, 72 hrs with E67, UNC 0638 or DMSO at concentrations ranging from 0.125 μM-10 μM and 0.125 μM -4 μM, respectively. MTT assays were performed as described previously (5 (link),7 (link),24 (link)). Absorbance at 570 nm and background at 650 nm were read on a multiwell spectrophotometer. Results were expressed as a percentage (%) of the control (DMSO).

Two human RAS mutated non-small cell lung adenocarcinoma cell lines were used in this study. Both cell lines were purchased from American Type Culture Collection (ATCC, USA). All of these cell lines were authenticated by Integrative Genomics Core by using short tandem repeat polymorphism analysis. H1299 (p53-Null, N-RAS mutation) was cultured in RPMI 1640 (ATCC, USA) medium and A549 (p53-Wild-type, K-RAS mutation) was cultured with DMEM/F12 (Corning, USA) medium. For all cell lines, cell authentic growth medium was supplemented with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin. Cell lines were tested Mycoplasma free using colorimetric mycoplasma detection assay with HEK-Blue™-2 cells. Cells used for experiments were within 20 passages from thawing. UNC0638, a selective inhibitor for G9a and GLP methyltransferase, was purchased from Cayman Chemical Company (USA) and used at a concentration of 2.5 μM and 5 μM. Defactinib hydrochloride, a FAK inhibitor which inhibits FAK phosphorylation at Tyr397, was purchased from MedChemExpress (USA) and used at a concentration of 1μM. Parthenilide, NF-κB inhibitor, was purchased from Abcam and used at a concentration of 20 μM.