Autocrine RTK signaling in BCa cell lines.

(A) Left: Axl kinase activation (phospho-Axl/total-Axl protein, pAxl/tAxl), as determined by 2-site immunoassay, in serum-deprived J82 cells treated with Gas6 (5.7 nM, 1 h at 37°C) in the presence or absence of cabozantinib (cabo; 300 nM), relative to untreated cells. Maximum possible Axl kinase activation was measured by adding ATP during the immunoassay (20 micromolar, 20 min at 25°C) of lysates prepared from untreated J82 cells. Right top: Axl kinase activation (pAxl/tAxl) in serum-deprived J82 cells that had been transfected with GAS6 siRNA or scrambled (control) siRNA. In both panels, values are the mean +/- SD from triplicate samples; asterisks indicate significant difference from untreated control (p < 0.05). Right bottom: ethidium bromide visualization of PCR products specific for GAS6 mRNA (upper panel) vs 18S rRNA (lower panel) resolved by agarose gel electrophoresis. (B) Left: Akt kinase activation (pAkt/tAkt), as determined by 2-site immunoassay, in serum-deprived J82 cells treated with Gas6 (5.7 nM, 1 h at 37°C) in the presence or absence of cabozantinib (cabo; 300 nM), relative to untreated cells. Right: Akt kinase activation (pAkt/tAkt) in serum-deprived J82 cells that had been transfected with Gas6 siRNA or scrambled (control) siRNA. In both panels, values are the mean +/- SD from triplicate samples; asterisks indicate significant difference from untreated control (p < 0.05). (C) Left: Erk kinase activation (pErk/tErk), as determined by 2-site immunoassay, in serum-deprived J82 cells treated with Gas6 (5.7 nM, 1 h at 37°C) in the presence or absence of cabozantinib (cabo; 300 nM), relative to untreated cells. Right: Erk kinase activation (pErk/tErk) in serum-deprived J82 cells that had been transfected with GAS6 siRNA or scrambled (control) siRNA. In both panels, values are the mean +/- SD from triplicate samples; asterisks indicate significant difference from untreated control (p < 0.05). (D) Upper panels: Immunoblot (IB) of phospho-MST1R (pMST1R) and MST1R (left) or pCSF1R and CSF1R (right) in lysates from serum-deprived FL3 cells after transfection with siRNA directed against MST1 (siMST1, left) or CSF1 (siCSF1, right) or control siRNA (siCont). Lower panels: Ethidium bromide visualization of PCR products (PCR) specific for MST1 (left) or CSF1 (right) mRNA or 18S RNA resolved by agarose gel electrophoresis. (E) Left: Akt kinase activation (pAkt/tAkt) in serum-deprived FL3 cells treated with MST1 (5 nM) or CSF1 (10 nM) in the presence or absence of cabozantinib (cabo; 300 nM), relative to untreated cells. Right: Akt kinase activation (pAkt/tAkt) in FL3 cells that had been transfected with siRNA directed against CSF1 or MST1, or with control siRNA. In both panels, values are the mean +/- SD from triplicate samples; asterisks indicate significant difference from control (p < 0.05). (F) Left: Erk kinase activation (pErk/tErk) in serum-deprived J82 cells treated with MST1 or CSF1 in the presence or absence of cabozantinib (cabo), relative to untreated cells. Right: Erk kinase activation (pErk/tErk) in FL3 cells that had been transfected with siRNA directed against CSF1 or MST1, or with control siRNA. In both panels, values are the mean +/- SD from triplicate samples; asterisks indicate significant difference from untreated control (p < 0.05). All results are representative of at least 3 experiments.