Figure 6. (A) AXL promotes resistance to BRAF inhibitor in PTEN wildtype melanoma cells.
(Top panel) The western blotting showed that the protein level of AXL in WM88 cells transfected with empty vector (EV), ALX wildtype (WT) or AXL kinase-dead mutant form (DN). (bottom panel) Cell viability curves of WM88 cells transfected with empty vector (EV), expressing AXL wildtype (WT) or AXL kinase-dead mutant form (DN) plasmid in response to PLX4032 were determined using CCK8 assay. Error bars denote s.d. for biological three repeats. (B) The constitutively active Akt abrogates the inhibition of PLX-4032 resistance mediated by shAXL. (Top panel) The western blotting showed that the protein level of AKT in WM88R/shAXL cells transfected with empty vector (EV), expressing Myr-AKT1 (AKT1), Myr-AKT2 (AKT2) or Myr-AKT3 (AKT3) plasmid. (bottom panel) Cell viability curves of WM88R/shAXL cells transfected with empty vector (EV), expressing Myr-AKT1 (AKT1), Myr-AKT2 (AKT2) or Myr-AKT3 (AKT3) plasmid in response to PLX4032 were determined using CCK8 assay. Error bars denote s.d. for biological three repeats. (C, D, and E) A pharmacological inhibitor R428 of AXL decreases the phosphorylation of AKT and restores the sensitivity of WM88R and A375smR cells to BRAF inhibitor with synergistic inhibition. (C) The expression of indicated proteins in WM88R and A375smR cells treated with 2 μM AXL inhibitor R428 and 2 μM BRAF inhibitor PLX4032 for 2 hours was determined by western blot. (D) The cell viability of WM88R and A375smR treated with BRAF inhibitor, or combination of BRAF inhibitor and AXL inhibitor R428 was determined by CCK8 assay. Error bars denote s.d. for biological three repeats. (E) Combination indexes (CI) for WM88R or A375smR cells treated with the combination of PLX4032 and R428 were determined using the Chou and Talalay method.