Fig. 6

A Western blotting for EGFR and AXL expressions in lysates from OC cell lines representative of HGSOCs or non-HGSOCs cell lines [34]. SKOV3 and OVCAR5 cells co-expressed both EGFR and AXL. B Confocal immunofluorescence showing EGFR and Axl expressions; only SKOV3 showed both EGFR (green) and AXL (red) expressions on the cell membrane with several regions of co-localization (white box indicates one of those regions). In OVCAR5 cells only EGFR is clearly expressed on the membrane. SKOV3 was chosen for further analysis. Upper, merge staining; lower. single staining, Nuclei were stained with DAPI. C upper. Western blotting of lysated from SKOV3 cells stimulated with GAS6 or EGF alone or in combination; lower, densitometric analysis for phoshorylated (P-EGFR and P–AXL) or total RTKs (EGFR and AXL). As expected, the total amount of EGFR decreased upon ligand stimulation [35]. No changes in the amount of AXL were detected upon GAS6 stimulation. D Erlotinib susceptibility of AXL silenced SKOV3 cells. Upper, western blotting showing the amount of silenced AXL in SKOV3 lysates upon transfection with two different siRNAs (#1 and #2). Lower, viability of SKOV3 cells treated with control siRNA (siCO) or with specific Axl siRNAs (siAxl#1 and #2) and then treated with erlotinib at different concentration. Statistical evaluation by ANOVA, p ≤ 0.001. Refer to Methods section for detailed procedure. The table below reports the IC50 values of siRNA transfected cells. The experiments were performed at least three times