Perhexiline maleate salt

Hepatocellular carcinoma cell line HepG2 was obtained from DSMZ (DSMZ, Braunschweig, Germany) and cultivated according to DSMZ instructions. A proliferation assay was performed using the colorimetric CellTiter 96^® AQ_ueous One Solution Cell Proliferation Assay (MTS) (Promega, Fitchburg, USA) according to instructions from the manufacturer. In brief, HepG2 was separately treated with 2 and 4 μM of sorafenib (Santa Cruz Biotechnology, Inc., Dallas, USA) and with 2, 4, 8, and 20 μM of perhexiline maleate salt (Sigma‐Aldrich, St. Louis, USA) for 24 and 48 h. Both compounds were dissolved in DMSO, and corresponding concentrations of DMSO in the medium were used as controls. In addition, controls consisting of cells growing in only cell culture medium were included. For all concentrations of sorafenib and perhexiline maleate salt, and for corresponding DMSO controls, eight replicates were analyzed. For the controls consisting of cells in only medium, 16 replicates were analyzed. For all experiments, the measured colorimetric differences between DMSO controls and medium controls were insignificant.

Perhexiline maleate salt was purchased from Sigma-Aldrich (St. Louis, MO, USA). Neuregulin was purchased from R&D Systems (Minneapolis, MN, USA). The following antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA): HER2 (3B5), HER3 (C-17), and ubiquitin. The pAkt (Ser473), pERK1/2, total EGFR, and Alexa Fluor™ 488 Conjugate Flag antibodies were purchased from Cell Signaling (Beverly, MA, USA). The antibodies were used at a 1:500 dilution in Western blotting. LysoTracker™ Red DND-99 was purchased from Invitrogen (Grand Island, NY, USA).