Additional file 6 of Gossypetin ameliorates 5xFAD spatial learning and memory through enhanced phagocytosis against Aβ

Additional file 6: Fig.S1 Gossypetin does not affect expression of β-, and γ-secretases and activity of β-secretase. (A to G) Time dependent β-secretase activity of mouse hippocampal lysate was measured with Relative Fluorescence Unit (RFU). Fluorescence excitation and emission wavelength was 335 nm and 495 nm respectively (A). Bar graph of RFU at each time point of 10 min (B), 20 min (C), 30 min (D), 40 min (E), 50 min (F), 60 min (G). (n = 10~12 mice per group) (H to L) Representative images of Western blot analysis for β-, γ-secretase subunits, and GAPDH (H). Bar graphs represent relative protein expression levels of BACE1 (I), Nicastrin (J), APH-1 (K), and PEN2 (L). (n = 12~15 mice per group) (M to P) Bar graphs represent relative mRNA expression level of β-, and γ-secretase subunits bace1 (M), ncstn (N), aph1 (O), pen2 (P). (n = 9~10 mice per group) Error bars represent the mean ± SD, *p < 0.05, ns = not significant, two-way ANOVA followed by Tukey’s multiple comparisons test. Fig. S2 Cell type classification of brain samples. (A) UMAP plot showing all cells from the brain samples, colored by their cell types. (B) Heatmap illustrating the Z-scores of average normalized expressions of cell type markers. (C) Violin plots displaying the log-scaled number of detected genes (top), Unique Molecular Identifiers (UMIs) (middle), and the percentage of mitochondrial gene expressions (bottom) per cell for each cell type. (D) UMAP plots showing all cells from the brain samples, colored by their sampled region (left), mouse strain (middle), or drug administration (right) condition. Fig. S3 Detailed subtyping of the microglial population. (A) UMAP plots showing all microglial cells from cortex region. The cells are colored by their celltypes (left). Heatmap showing the Z-scores of average normalized expressions of representative DEGs for each cell type from cortex region (right). (B) UMAP plots showing microglial cells from cortex (left) or hippocampus (right), colored by combination of mouse strain and drug administration condition. (C) UMAP plots illustrating microglial cells from cortex (left) or hippocampus (right), colored by their inferred cell cycle. (D) Bar plots for the fraction of cortex (left) or hippocampus (right) microglial cells by sample conditions, which are the combination of mouse strain and drug administration, for each microglial subtype. Fig. S4 Differential gene expressions between vehicle- and gossypetin-treated microglia. (A) Scatter plot showing GOBP terms that are upregulated or downregulated by5xFAD construction or gossypetin administration for each microglial subtype from cortex. Significant (Fisher’s exact test, P < 0.01) terms associated with antigen presentation are colored by their biological keywords. (B) GSEA plots showing significant (P< 0.05) GOBP terms for gossypetin administration condition against vehicle treatment within 5xFAD homeostatic microglia from hippocampus region. Related to Fig. 3D. (C) Volcano plot illustrating the DEGs selected by the comparison between wild type and 5xFAD(left), or vehicle and gossypetin treated 5xFAD (right) from homeostatic microglial population of cortex region. Fig. S5 Transcriptomic transition in cortex microglia and measurement of DAM signature score. (A) Volcano plot showing significant (p < 0.05) DEGs selected by the comparison between cortex homeostatic microglia in vehicle treated wild type and 5xFAD (top left), or vehicle and gossypetin treated 5xFAD (top right). Volcano plots illustrating comparison between gossypetin administration condition against vehicle treatment within 5xFAD stage 1 DAM (bottom left) or stage 2 DAM (bottom right) from cortex are also presented. (B) Violin plot illustrating module scores for the DAM-related genes from previous studies. Cells are grouped by the combination of their mouse strain and treatment condition. (***P < 0.001) Fig. S6 Gossypetin ameliorates gliosis in microglia and astrocytes. (A to D) Representative images of hippocampus (A) and cortex (C) stained with Hoechst and Iba-1. Scale bar corresponds to 200μm. Bar graph represents quantification of Iba-1 positive area in dentate gyrus of hippocampus (n = 9~12 mice per group, 3~6 slices per brain) (B) and cortex (n = 9~12 mice per group, 3~6 slices per brain) (D). (E to H) Representative images of hippocampus (E) and cortex (G) stained with Hoechst and GFAP. Scale bar corresponds to 200μm. Bar graph represents quantification of GFAP positive area in dentate gyrus of hippocampus (n = 9~12 mice per group, 3~6 slices per brain) (F) and cortex (n = 9~12 mice per group, 3~5 slices per brain) (H). The error bars represent the mean ± SEM.****p <0.0001, ***p < 0.001, **p < 0.01, ns = not significant, two-way ANOVA followed by Tukey’s multiple comparisons test (B, D, F and H). Fig. S7 Gossypetin increases Aβ phagocytic capacity and dynamics of BV2 microglial cell line. (A) Representative images of BV2 cells treated with 488-Aβ and stained with Hoechst and Iba-1. Gossypetin (25μM) was pretreated for 24 h before 488-Aβ treatment. Scale bar corresponds to 100μm. (B). Bar graph represents quantification of area of internalized 488-Aβ in BV2 (n= 3 per group, 253~656 cells per sample). (C) Line graph represents quantification of fluorescent area generated by internalized 488-Aβ in BV2 in a time dependent manner (n = 3 per group, 107~347 cells per sample). The error bars represent the mean ± SEM. ****p <0.0001, *p < 0.05, two-way ANOVA followed by Tukey’s multiple comparisons test (C), Student’s t test (B).