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).

附加文件6：图S1 桑黄素（gossypetin）不影响β-分泌酶（β-secretase）与γ-分泌酶（γ-secretase）的表达，以及β-分泌酶的活性。(A至G) 采用相对荧光单位（Relative Fluorescence Unit, RFU）检测小鼠海马裂解物的β-分泌酶活性随时间的变化情况。荧光激发与发射波长分别为335 nm与495 nm（A）。分别展示10 min（B）、20 min（C）、30 min（D）、40 min（E）、50 min（F）、60 min（G）各时间点的RFU柱状图。(每组n=10~12只小鼠)

(H至L) β-、γ-分泌酶亚基及甘油醛-3-磷酸脱氢酶（GAPDH）的蛋白质印迹（Western blot）分析代表性图像（H）。柱状图分别代表BACE1（I）、Nicastrin（J）、APH-1（K）及PEN2（L）的相对蛋白表达水平。(每组n=12~15只小鼠)

(M至P) β-、γ-分泌酶亚基bace1、ncstn、aph1及pen2的相对mRNA表达水平柱状图。(每组n=9~10只小鼠)

误差棒表示平均值±标准差（standard deviation, SD），*p < 0.05，ns=无显著性差异，采用双因素方差分析（two-way ANOVA）后进行Tukey多重比较检验。

图S2 脑组织样本的细胞类型分类。(A) UMAP（Uniform Manifold Approximation and Projection）图展示脑组织样本的所有细胞，按细胞类型着色。(B) 热图展示细胞类型标志物的平均归一化表达量的Z分数。(C) 小提琴图分别展示每种细胞类型的每个细胞的检测基因数（上）、唯一分子标识符（Unique Molecular Identifiers, UMIs，中间）及线粒体基因表达占比（下）的对数缩放值。(D) UMAP图展示脑组织样本的所有细胞，分别按取样区域（左）、小鼠品系（中）或给药条件（右）着色。

图S3 小胶质细胞群体的精细分型。(A) UMAP图展示来自皮层区域的所有小胶质细胞，按细胞类型着色（左）。热图展示皮层区域各细胞类型的代表性差异表达基因（Differentially Expressed Genes, DEGs）的平均归一化表达量的Z分数（右）。(B) UMAP图分别展示来自皮层（左）或海马（右）的小胶质细胞，按小鼠品系与给药条件的组合着色。(C) UMAP图分别展示来自皮层（左）或海马（右）的小胶质细胞，按预测的细胞周期状态着色。(D) 柱状图展示每种小胶质细胞亚型按样本条件（即小鼠品系与给药条件的组合）划分的皮层（左）或海马（右）小胶质细胞占比。

图S4 溶剂对照组与桑黄素处理组小胶质细胞间的差异基因表达。(A) 散点图展示皮层各小胶质细胞亚型中，由5xFAD造模或桑黄素给药所上调或下调的基因本体生物过程（Gene Ontology Biological Process, GOBP）术语。与抗原呈递相关的显著（Fisher精确检验，P < 0.01）术语按其生物学关键词着色。(B) 基因集富集分析（Gene Set Enrichment Analysis, GSEA）图展示来自海马区域的5xFAD稳态小胶质细胞中，桑黄素给药组相较于溶剂对照组的显著（P<0.05）GOBP术语。与图3D相关。(C) 火山图展示分别来自皮层稳态小胶质细胞群体的两组比较：野生型与5xFAD小鼠（左），以及溶剂处理与桑黄素处理的5xFAD小鼠（右）所筛选出的差异表达基因。

图S5 皮层小胶质细胞的转录组转变及疾病相关小胶质细胞（Disease-Associated Microglia, DAM）特征评分检测。(A) 火山图分别展示：溶剂处理的野生型与5xFAD小鼠皮层稳态小胶质细胞的比较（左上），以及溶剂处理与桑黄素处理的5xFAD小鼠皮层稳态小胶质细胞的比较（右上）。同时展示来自皮层的5xFAD阶段1 DAM（左下）或阶段2 DAM（右下）中，桑黄素给药组相较于溶剂对照组的比较结果。(B) 小提琴图展示来自既往研究的DAM相关基因的模块评分。细胞按小鼠品系与处理条件的组合分组。(***P < 0.001)

图S6 桑黄素改善小胶质细胞与星形胶质细胞的胶质增生。(A至D) 用Hoechst与离子钙接头蛋白分子1（ionized calcium-binding adapter molecule 1, Iba-1）染色的海马（A）及皮层（C）代表性图像。比例尺为200μm。柱状图分别代表海马齿状回（B）及皮层（D）中Iba-1阳性区域的定量结果(每组n=9~12只小鼠，每脑取3~6张切片)。(E至H) 用Hoechst与胶质纤维酸性蛋白（glial fibrillary acidic protein, GFAP）染色的海马（E）及皮层（G）代表性图像。比例尺为200μm。柱状图分别代表海马齿状回（F）及皮层（H）中GFAP阳性区域的定量结果(每组n=9~12只小鼠，每脑取3~5张切片)。误差棒表示平均值±标准误（standard error of the mean, SEM）。****p <0.0001，***p < 0.001，**p < 0.01，ns=无显著性差异，采用双因素方差分析后进行Tukey多重比较检验（B、D、F及H）。

图S7 桑黄素增强BV2小胶质细胞系的Aβ吞噬能力与吞噬动力学。(A) 用488标记的淀粉样β蛋白（amyloid β-protein, Aβ）处理、并经Hoechst与Iba-1染色的BV2细胞代表性图像。桑黄素(25μM)于488-Aβ处理前预孵育24 h。比例尺为100μm。(B) 柱状图代表BV2细胞中内化的488-Aβ区域的定量结果(每组n=3，每个样本计数253~656个细胞)。(C) 折线图代表BV2细胞中内化的488-Aβ产生的荧光面积随时间的定量结果(每组n=3，每个样本计数107~347个细胞)。误差棒表示平均值±标准误。****p <0.0001，*p < 0.05，分别采用双因素方差分析后进行Tukey多重比较检验（C）及Student t检验（B）。