Figure 4: Combined treatment with UNC2025 and a BRAF or MEK inhibitor enhances functional anti-tumor effects in BRAF^mt and NRAS^mt melanoma cells.

A) Serum starved BRAF^mt G361 cell cultures were treated with 300 nM UNC2025 and/or 675 nM vemurafenib or DMSO vehicle for 90 min and then stimulated with 200 nM GAS6 ligand or an equivalent volume of vehicle for an additional 10 min. Cell lysates were prepared and the indicated phosphorylated (denoted by p-) and total proteins were detected by immunoblot. B) G361 and A101D cell lines were cultured at low density and treated with 1μM vemurafenib alone or in combination with the indicated concentrations of UNC2025, or with vehicle (DMSO) only for 10 days. Colonies were stained with crystal violet and enumerated. Colony numbers relative to vehicle treated cultures are shown. C) G361 and A101D cell cultures were treated with 1μM vemurafenib alone or in combination with the indicated concentrations of UNC2025 or with vehicle (DMSO) only for 48 h and then stained with PO-PRO^™−1 iodide (PO-PRO) and 7-AAD. Apoptotic (PO-PRO+, 7-AAD negative) and dead (7-AAD+) cells were detected by flow cytometry. Dead and dying cells were enumerated via flow cytometric analysis based on PO-PRO™-1 and 7-AAD uptake. D&E) HMCB cell cultures were treated with the indicated concentrations of cobimetinib (cobi), UNC2025, the combination, or vehicle and colony formation (D) and induction of cell death (E) were assessed as above. n=3–4 independent experiments, *p < 0.05, **p < 0.01, ***p<0.001, one-way ANOVA.