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Figure S1. Morphology of microglial cells in neuron-microglia co-cultures. CD11b immunostaining in control (a) and LPS/IFN-γ (b), chrysin (Ch) (c) and Ch p;+ p;LPS/IFN-γ (d) treated co-cultures. CD11b immunostaining was performed 48 p;h after LPS/IFN-γ treatment. Insets in (a) and (b) show microglial cells at a higher magnification. Control microglia show a weak labelling in most of the cell surface, and a strong labelling at the end of some processes. Notice the morphological change and the increase in CD11b immunostaining in most of the microglial cell surface in LPS/IFN-γ-treated co-cultures, both in the absence and in the presence of chrysin pre-treatment. Bars p;= p;20 p;μm (insets) and 50 p;μm.

Figure S2. Anti-inflammatory effects of chrysin in BV2 cells. Effect of chrysin (Ch) pre-treatment on (a) NO and (b) TNF-α production, and (c) iNOS and (d) COX-2 protein expression in control and in LPS/IFN-γ, chrysin (Ch) and Ch p;+ p;LPS/IFN-γ treated cells. Measurements were performed after 24 p;h (NO and TNF-α production) or 12 p;h (iNOS and COX-2 expression) of LPS/IFN-γ treatment. Protein expression was determined by western blot and data normalized with ß-actin expression. Bars represent means p;± p;SEM of three to four independent experiments. *p p;< p;0.05, **p p;< p;0.01 and ***p p;< p;0.001 vs. control; #p p;< p;0.05; ##p p;< p;0.01 vs. LPS/IFN-γ; one-way anova (repeated measures) and Newman–Keuls post-test. (e) Images show representative western blots.

Figure S3. Neuroprotective action of chrysin pre-treatment in neuronal-BV2 co-cultures treated with LPS/IFN-γ. MAP2 immunostaining in control (a) and LPS/IFN-γ (b), chrysin (Ch) (c) and Ch p;+ p;LPS/IFN-γ (d) treated co-cultures. MAP2 immunostaining was performed 24 p;h after treatment. Arrowheads in (a) point out MAP2 immunostaining in neuronal processes, which are abundant in (a), (c) and (d) and dramatically decreased in (b), what is taken as an index of neuronal damage/death. Bar p;= p;100 p;μm. (e) Evaluation of neuronal viability 24 p;h after treatment by MAP2-ABTS-ELISA assay. Results are presented as % of MAP2 immunostaining in control cultures. (f) NO and (g) TNF-α production in control, LPS/IFN-γ, and Ch p;+ p;LPS/IFN-γ treated co-cultures. Bars represent means p;± p;SEM of four independent experiments. *p p;< p;0.05, **p p;< p;0.01 and ***p p;< p;0.001 vs. control; #p p;< p;0.05, ##p p;< p;0.01 and ###p p;< p;0.001 vs. LPS/IFN-γ; one-way anova (repeated measures) and Newman–Keuls post-test.

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