Additional file 1 of BACE1 regulates expression of Clusterin in astrocytes for enhancing clearance of β-amyloid peptides

Additional file 1: Supplemental Figure S1. Quality control measure of Bace1-/- and Bace1+/+ scRNAseq. (A) Violin plots of data set quality control measurement for nFeature_RNA, nCount_RNA, and percent.mt generated from each ACSA2+-enriched sample. Samples 1a, 2a, 3a are from Bace1+/+ mice, while samples 4a, 5a, 6a are from Bace1-/-. Filtered cutoff points were set at GEMS containing >1,000 identified genes, <25,000 read counts, and <20% mitochondrial RNA. (B) Visualization of UMAP dimension plot with identified cell type clusters from Bace1-/- and Bace1+/+ scRNAseq. R Astrocytes refers reactive astrocytes, and OPC stands for oligodendrocyte precursor cells. The visible difference is the increase in the R Astrocyte cluster in Bace1-/- samples. Supplemental Figure S2. Quality control measure of 5xFAD;Bace1fl/fl;UBC-creER and 5xFAD;Bace1fl/fl. Quality control measured for nFeature_RNA, nCount_RNA, and percent.mt generated from pooled ACSA2+-enriched samples from 5xFAD;Bace1fl/fl;UBC-creER (Sample 1A) and 5xFAD;Bace1fl/fl (Sample 3a) Filtered cutoff points were set at GEMS containing >1,000 identified genes, <25,000 read counts, and <20% mitochondrial RNA. Supplemental Figure S3. Validation of siRNA Clu knockdown. (A) Western blot of WT primary astrocytes treated with either 80, 40, 20, 10 pmol of Clu siRNA or 80 pmol of control scrambled siRNA. Images indicate major bands for CLU and actin. (B) CLU band intensity normalized to actin. We noted that 80 pmol of Clu siRNA resulted in an approximately 50% decrease in LU levels compared to control siRNA. Supplemental Figure S4. Targeted astrocytic deletion of Bace1 increases Aβ clearance. Representative images from Thioflavin-S staining of amyloid plaques from fixed saggital brain sections of 5xFAD;Bace1fl/fl;Gfap-cre and 5xFAD;Bace1fl/fl. Insets highlight hippocampal and cortical regions that are presented in Fig. 9A. Supplemental Table 1. List of differentially expressed genes of Bace1-/- reactive astrocytes. Supplemental Table 2. List of differentially expressed genes of 5xFAD;Bace1fl/fl;UBC-creER reactive astrocytes.

附加文件1：补充图S1。Bace1基因敲除（Bace1-/-）与野生型（Bace1+/+）单细胞RNA测序（scRNAseq）的质量控制检测。(A) 针对每份ACSA2+富集样本生成的nFeature_RNA、nCount_RNA及percent.mt三项质量控制指标的小提琴图。其中样本1a、2a、3a取自Bace1+/+小鼠，样本4a、5a、6a取自Bace1-/-小鼠。筛选 cutoff 值设定为：基因表达矩阵（GEMS）中检测到的基因数>1000、读取计数<25000、线粒体RNA占比<20%。(B) Bace1-/-与Bace1+/+ scRNAseq的UMAP降维可视化图，标注了鉴定出的细胞类型簇。其中R Astrocytes指反应性星形胶质细胞，OPC为少突胶质前体细胞（oligodendrocyte precursor cells）。可见差异为Bace1-/-样本中的反应性星形胶质细胞簇占比升高。

补充图S2：5xFAD;Bace1fl/fl;UBC-creER与5xFAD;Bace1fl/fl样本的质量控制检测。针对来自5xFAD;Bace1fl/fl;UBC-creER（样本1A）与5xFAD;Bace1fl/fl（样本3a）的混合ACSA2+富集样本，生成nFeature_RNA、nCount_RNA及percent.mt三项质量控制指标。筛选cutoff值设定为：GEMS中检测到的基因数>1000、读取计数<25000、线粒体RNA占比<20%。

补充图S3：Clu基因小干扰RNA（siRNA）沉默效率验证。(A) 用浓度分别为80、40、20、10 pmol的Clu siRNA，或80 pmol的阴性对照乱序siRNA处理野生型原代星形胶质细胞的蛋白质免疫印迹（Western blot）结果。条带分别对应CLU蛋白与肌动蛋白（actin）。(B) CLU蛋白条带灰度值经肌动蛋白归一化后的结果。结果显示，80 pmol的Clu siRNA可使CLU蛋白表达量较阴性对照组降低约50%。

补充图S4：星形胶质细胞特异性敲除Bace1可增强Aβ清除能力。展示5xFAD;Bace1fl/fl;Gfap-cre与5xFAD;Bace1fl/fl小鼠固定矢状脑切片的硫黄素-S（Thioflavin-S）染色淀粉样斑块代表性图像。插图标注了与图9A一致的海马区与皮层区域。

补充表1：Bace1-/-反应性星形胶质细胞的差异表达基因列表。

补充表2：5xFAD;Bace1fl/fl;UBC-creER反应性星形胶质细胞的差异表达基因列表。