Fig. 4. Involvement of TRKB/TGFß-RII and TGFß2 signaling pathways in the anti-mitotic and differentiating effects of CM_Cerebellum. (A) Transfection of cells with a dominant-negative form () of TRKB (TRKB) or TGFß receptor II (TGFß-RII) before treatment with CM_Cerebellum resulted in a significant increase in proliferation (versus controls transfected with GFP) (100% refers to BrdU incorporation in absence of CM_Cerebellum). (B) Results from cells treated as described above show that inhibition of expression of TGFß-RII or TRKB prevents CM_Cerebellum-induced neuronal differentiation, as measured by number of MAP2A/MAP2B-positive cells (cells not exposed to CM_Cerebellum provide the reference value of 100%). (C) Exposure of hippocampal cells to CM_Cerebellum, as well as to either TGFß2 (1 ng/ml) or BDNF (100 ng/ml) induces nuclear translocation of the TGFß2-specific partner SMAD2 and of co-SMAD4 within 3 hours, as shown by transient transfection experiments. (D,E) Introduction of dominant-negative forms of either SMAD3 (which specifically couples with TGFß2) or of Co-SMAD4 abrogates the anti-mitotic (D) and differentiating (E) effects of CM_Cerebellum on primary hippocampal cells; these changes do not reflect apoptosis as the number of TUNEL-stained and activated caspase 3 cells were not altered by the treatment (data not shown). (F-H) CM_Cerebellum stimulates generation of luciferase from the TGFß reporter gene 3TP-Lux in HiB5 cells (F), an effect abrogated when expression of either SMAD3 or Co-SMAD4 is blocked by transfection with their respective dominant-negative () forms (G); similarly, transactivation of 3TP-Lux is blocked in the presence of TGFß-RII and TRKB (H). Each half set of data in A,B,D,E were obtained in independent experiments in which GFP was used to control for between-culture variability in transfection efficiency. Data in G,H represent ratios of luciferase expression (fold change in treatments versus non-treated cells). In all cases, transfections were performed 24 hours before addition of CM_Cerebellum, TGFß2 or BDNF for 24 hours, after which the analysis was performed. Numerical data refer to mean±s.d. (n=4-6) *P<0.05, **P<0.01, ***P<0.001 (versus appropriate controls).