Fig. 6

EGFR and RYK sustain progranulin signaling in mesothelioma cells. A Total and phosphorylated EphA2 (S897), AKT and ERK1/2 in EphA2 KO MSTO-211H cells stably transduced with an empty vector or re-expressing wild type EphA2 (left panel) and NCI-H2052 cells (right panel) pre-incubated with the EGFR inhibitor gefitinib (10 µM) for 1 h and then exposed to the same concentration of gefitinib alone or in combination with 50 nM progranulin for 15 min. B Total and phosphorylated EGFR (Y1068) in MSTO-211H cells transfected with GRN-specific or non-targeting control (siCTR) siRNA (upper left panel), parental and PGRN overexpressing NCI-H2052 cells (upper right panel) and parental, GRN KO or PGRN-reconstituted GRN KO MSTO-211H cells (lower panel) after serum-starvation for 24 h. C Levels of total and phosphorylated EGFR (Y1068), EphA2 (S897) and AKT in parental or GRN KO MSTO-211H cells serum-starved for 24 h and treated with gefitinib (10 µM) for 1 h 30 min. D Levels of EphA7, total and phosphorylated EphA2 (S897), AKT and ERK1/2 in MSTO-211H cells transfected with EphA7-specific or control (siCTR) siRNA, transferred onto serum-free 24 h post transfection, incubated for additional 24 h and then treated with 50 nM progranulin for 15 min. E MSTO-211H and NCI-H2052 cells were transfected with siRNA specific for RYK or non-targeting control (siCTR) siRNAs. At 48 h post-transfection cells were transferred onto serum-free medium and incubated for 24 h. Cells were then treated with 50 nM PGRN for 15 min. Total and phosphorylated EphA2 (S897), AKT and ERK1/2 were analyzed by immunoblot. F-G Total and phosphorylated AKT and ERK1/2 in NCI-H2052 transfected with siRYK or control siRNA (siCTR) (F) or stably expressing two different RYK-specific or a non-targeting control (shCTR) shRNA treated with 10 µM gefitinib for 1 h and then exposed to the same concentration of gefitinib alone or in combination with 50 nM progranulin for 15 min