Figure 1.

BafA1 treatment modulates aggregate formation and levels of extracellular SNCA in transfected H4 cells. (A) to (C): Immunocytochemistry of H4 cells transfected with SNCA, SNCA-T and SNCA-T and SNCAIP: (A) Large aggregates of different size and morphology can be observed after transfection with high-aggregating SNCA-T and SNCAIP whereas SNCA only occasionally leads to smaller intracellular aggregates sized below 1 μm. Scale bar: 5 μm. (B) Quantification of differentially sized aggregates per cell in H4 cells transfected with SNCA-T alone compared to cotransfection of SNCA-T with SNCAIP. (C) Treatment with 200 nM BafA1 for 12 h results in smaller and more punctate intracellular structures. (D) to (F): Dot blot analysis of extracellular SNCA in the conditioned medium of H4 neuron-like cells and CG4 oligodendroglial cells: (D) Representative dot blots of H4 cell medium containing extracellular SNCA. Recombinant SNCA is used as quantification standard. (E) Dot blot quantification of extracellular SNCA levels in similar volumes of conditioned medium of H4 cells expressing high-aggregating SNCA-T compared to low-aggregating SNCA and mock-transfected control cells 36 h post-transfection. Treatment with 200 nM BafA1 for 12 h results in slightly increased levels of extracellular SNCA in the medium of H4 cells transfected with SNCA, and both SNCA-T and SNCAIP compared to untreated cells. (F) Representative dot blots showing a lack of extracellular SNCA in supernatants of undifferentiated and 6 d differentiated rat oligodendroglial CG4 cells overexpressing SNCA to exclude nonspecific SNCA release. All values are presented as mean + s.e.m; (*) P = 0.009.