Fig. 5. Cytotoxicity of E67-2 compounds to primary fibroblasts.

Mouse and human primary fibroblasts were treated with various concentrations of BIX-01294, E67, and E67-2. Primary mouse embryonic fibroblasts (MEFs) and mouse tail-tip fibroblasts (MTTFs) were isolated from E13.5 embryo and tail, respectively, from 129S2/SvPasCrl inbred mouse (Charels River, Wilmington, MA, USA). Human embryonic fibroblasts (HEFs), HE-19, was purchased from RIKEN Cell Bank (Ibaraki, Japan). Human dermal fibroblasts (HDFs) were purchased from American Type Culture Collection (ATCC, Washington, DC, USA). All primary cells (100 µl) were cultured in Dulbecco’s modified Eagle’s Media (DMEM, Invitrogen, Carlsbad, CA, USA) supplement with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA, USA), 1x Antibiotic-Antimycotic (Invitrogen, Carlsbad, CA, USA), 2 mM L-glutamine (GlutaMAX, Invitrogen, Carlsbad, CA, USA), 100 µM Minimum Essential Media Non-Essential Amino Acids (MEM NEAA, Invitrogen, Carlsbad, CA, USA) and 100 µM β-mercaptoethanol (Sigma, St. Louis, MO, USA). Cell cytotoxicity was measured with the cell counting kit-8 (CCK-8, Dojindo, Kumamoto, Japan). Five thousand cells in 100 µl were seeded per well. After incubation for 24 h at 37°C, 5% CO₂, cells were treated with BIX-01294, E67, and E67-2 at 100, 300, 1,000, 3,000, 10,000, 30,000, and 100,000 nM and incubated another 24 h. A 10 µl of CCK-8 solution was added to each well and the plate was incubated for another 4 h. Cell viability was determined with colorimetric assays for measuring the activity of dehydrogenase enzyme from live cells that reduce a chemical dye, WST-8, to an orange-color formazan dye which can be detected at a specific wavelength of light. The absorbance of formazan dye at 450 nm is directly proportional to the number of living cells. The absorbance was read at 450 nm using Synergy 2 microplate reader (BioTek, Winooski, VT, USA).