Fig. 3.

Progranulin activates AKT and ERK1/2 in an EphA2-independent manner in mesothelioma cells. A MSTO-211H cells were transfected with siRNA targeting either GRN (progranulin), EphA2, the combination of the two or non-targeting control (siCTR). At 8 h post-transfection cells were transferred to serum-free media and incubated for additional 40 h. Cells were then treated with 50 nM progranulin for 60 min. Levels of progranulin, total and phosphorylated EphA2 (S897), AKT and ERK1/2 were analyzed by immunoblot. B GRN was depleted using siRNAs targeting progranulin as described in (A) in parental MSTO-211H (P), EphA2 KO MSTO-211H and EphA2 KO cells re-expressing wild type EphA2 or the EphA2 mutants EphA2 K646M and EphA2 S897A/S899A/S901A. Levels of progranulin, total and phosphorylated EphA2 (S897), AKT and ERK1/2 were determined by immunoblot. C Total and phosphorylated EphA2 (S897), AKT and ERK1/2 as assessed by immunoblot in EphA2 KO MSTO-211H cells stably transfected with an empty vector or re-expressing wild-type EphA2, serum-starved for 24 h and exposed to 50 nM progranulin for the indicated time. D Parental (P) NCI-H2052 cells, two different EphA2 KO NCI-H2052 clones and a non-targeting control (NTC) NCI-H2052 clone were serum-starved for 24 h and then stimulated with 50 nM progranulin for 15 min. Levels of total and phosphorylated EphA2, AKT and ERK1/2 were analyzed by immunoblot