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. 2012 Oct 12;7(10):e47312. doi: 10.1371/journal.pone.0047312

Figure 3. Riluzole increases Smad2 linker phosphorylation at the cluster of serines and Smad3 linker phosphorylation at serine 204 through GSK3.

Figure 3

A. Riluzole increases Smad2 and Smad3 linker phosphorylation. 24 hours post seeding, C8161, UACC930, WM278, WM793 and 1205LU human melanoma cell lines were serum-starved for about 16 hours, and incubated in the absence (−) or presence (+) of 25 µM of riluzole for 9 hours. Immunoblots were performed using phosphoSmad2 (Ser245/250/255); phosphoSmad3 (Ser204); Smad2 and Smad3; GAPDH. Two exposures are presented for phosphoSmad2 (Ser245/250/255) to see the lower signals for WM278. B. LiCl treatment counteracts riluzole-induced Smad linker phosphorylation. After serum starvation, cells were incubated in the absence (−) or presence (+) of 25 µM riluzole either in the presence of NaCl (−) or LiCl (+) for 9 hours. Immunoblots were performed as in A. C. Treatment with the specific GSK3 inhibitor, CT99021, counteracts riluzole-induced Smad linker phosphorylation. After serum starvation, cells were incubated in the absence (−) or presence (+) of 25 µM riluzole either in the absence (−) or presence (+) of CT99021 (CT) for 9 hours. Immunoblots were performed as in A. D. GSK3α and GSK3β knock-down inhibits riluzole-induced Smad linker phosphorylation. WM793 melanoma cells were transfected with non targeting control siRNA (NT si) or GSK3α/β siRNA (GSK3α/β si), serum-starved and incubated with or without riluzole for 9 hours before protein extraction. Immunoblots were done as in A. E. GSK3β can phosphorylate the cluster of serines 245/250/255 in Smad2 and serine 204 in Smad3 in vitro. Recombinant GSK3β was used to phosphorylate GST-Smad2 and GST-Smad3 in a non radioactive reaction. The reaction products were analyzed by immunoblotting as in A. Ril: Riluzole.