Fig. 4.

Mixture effects on BDNF protein levels. NSCs were differentiated for 7 DIV, and then treated for 14d with single chemicals or three types of mixtures as described in Fig. 3 legend. (A) Graph shows BDNF total levels measured upon treatment with single chemicals or mixtures at all tested LOAEC-bdnf concentrations (see Table 2). (B) Representative immunocytochemical image (at 10x magnification, with 40x magnifications insets) of cells treated with BDNF-related mixtures (at LOAEC/2-bdnf concentrations) for 14d and stained for β-III-Tubulin (red) and BDFN (green). For the analyses, all samples were normalised to solvent control (0.1 % DMSO, Ctr) at the respective time point. Data are represented as mean ± S.E.M. of 3-4 biological replicates. (C) Heatmaps showing the contribution of individual chemicals to overall mixtures’ effects (i.e., dark green: high correlation, same effect directionality; dark yellow: low correlation, opposite effect directionality). (D) Correlation plot to compare mixture effects (size and colour of the circles corresponds to the level of correlation between pair-wise mixtures: i.e., the bigger and darker green, the higher the level of correlation). C and D were calculated according to Pearson and the correlation matrix was calculated considering the response upon exposure to LOAEC/2-, LOAEC/4- and LOAEC/8-bdnf and effect trend/directionality.