Additional file 1 of Translational profiling identifies sex-specific metabolic and epigenetic reprogramming of cortical microglia/macrophages in APPPS1-21 mice with an antibiotic-perturbed-microbiome

Additional file 1: SI. Figure 1. Confirmation of APP/PS1 and FLAG-EGFP-Rpl10a transgenes in APPPS1-21-CD11br mice. A. Genomic DNA from APPPS1-21-CD11br mice was used for PCR amplification using primer set specific for APP, PS1 transgenes. APPPS1-21 mice serve as positive control. B. Schematic to show the binding sites of various primer sets to confirm the presence of FLAG-EGFP-Rpl10a transgene and the amplicon sizes. 1% Agarose gel to show the amplified products. SI. Figure 2. Characterization of CD11b+ cells from CD11br TRAP mice. Representative immunofluorescent images showing co-staining of CD11b with GFP and Iba I from brain sections of WT-CD11br and APPPS1-21-CD11br transgenic mice (A), microglia specific P2ry12 (magenta) and Tmem119 (red) markers colocalized with CD11b+ (green) macrophage/microglia cells from brain sections of 7-wks-old WT-CD11br and APPPS1-21-CD11br male mice (B), microglia specific P2ry12 (red) colocalized with CD11b+ (green) and 3D6 (magenta) stained for amyloid plaques in APPPS1-21-CD11br mice (C). (D) GFP (green) immunostaining colocalized with microglia marker P2ry12 (magenta) in brain sections of WT-CD11br, APPPS1-21-CD11br mice and APPPS1-21 that doesn’t show the expression of GFP was used as a control. The top panel of 2B-D shows low magnification and the selected microglia cells indicated in the box are shown at higher in the bottom panel. SI. Figure 3. Microglia specific transcripts from pull-down of cortical lysates of CD11br mice 7-week mice. Cortical lysates were used for immunoprecipitation of CD11b+ microglia/macrophage ribosomes from WT and APPPS1-21-CD11br male and female mice treated with vehicle or ABX and then for mRNA purification. Primer sets for Tmem119 (A) and P2ry12 (B) were used for RT-PCR analysis to confirm the pull-down of RNA from CD11b+ cells were abundant in microglia. Cortical lysates from APPPS1-21 and non-Tg mice were used as controls to indicate the specificity of pull-down. To account for the pulldown of CD11b expressing macrophages, following the synthesis of cDNA, primers specific for Hexb, a bonafide microglia marker and CD163, a selective marker of perivascular macrophages were used for RT-PCR and the amplicons were resolved on 1% Agarose gel (C, a). The ΔCt (Ct [Gene]-Ct [Gapdh]) values from qPCR analysis using the same primer sets to indicate the pulldown of CD11b+ cells were enriched for microglia (C, b). SI. Figure 4. A. Number of protein quantifications for each group comparison identified by LFQ. B. Venn diagram representing the number of proteins found in all replicates of each group of male mice (RM, TMV, TMA) and female mice (RF, TFV, TFA). SI. Figure 5. Gene Ontology and functional enrichment analysis of DEPs in vehicle-treated male AD (APPPS1-21-CD11br) mice using ClueGo and CluePedia plugins of Cytoscape. GO Terms (Biological Process, Cellular component, Molecular function, Immune pathway) associated with the identified proteins. The most significant parent or child term per functional group (kappa score ≥ 0.4) is shown in the functional grouped network as a group title (a). Peptides associated with the same term are represented by a node. Node color represents the class that they belong. Mixed coloring means that the specific node belongs to multiple classes. Upregulated proteins are shown in red and downregulated are shown in blue. Edges show the association of the peptide with the terms. The thickness of the edge reflects the association significance. (b) Pie chart to indicate specific GO terms (% terms per group). SI. Figure 6. Gene Ontology and functional enrichment analysis of DEPs in ABX-treated male AD (APPPS1-21-CD11br) mice using ClueGo and CluePedia plugins of Cytoscape. GO Terms (Biological Process, Cellular component, Molecular function, Immune pathway) associated with the identified proteins. The most significant parent or child term per functional group (kappa score ≥ 0.4) is shown in the functional grouped network as a group title (a). Peptides associated with the same term are represented by a node. Node color represents the class that they belong. Mixed coloring means that the specific node belongs to multiple classes. Upregulated proteins are shown in red and downregulated are shown in blue. Edges show the association of the peptide with the terms. The thickness of the edge reflects the association significance. (b) Pie chart to indicate specific GO terms (% terms per group). SI. Figure 7. Gene Ontology and Functional enrichment analysis of DEPs in vehicle-treated female AD (APPPS1-21-CD11br) mice using ClueGo and CluePedia plugins of Cytoscape. GO Terms (Biological Process, Cellular component, Molecular function, Immune pathway) associated with the identified proteins. The most significant parent or child term per functional group (kappa score ≥ 0.4) is shown in the functional grouped network as a group title (a). Peptides associated with the same term are represented by a node. Node color represents the class that they belong. Mixed coloring means that the specific node belongs to multiple classes. Upregulated proteins are shown in red and downregulated are shown in blue. Edges show the association of the peptide with the terms. The thickness of the edge reflects the association significance. (b) Pie chart to indicate specific GO terms (% terms per group). SI. Figure 8. Gene Ontology and Functional enrichment analysis of DEPs in ABX-treated female AD (APPPS1-21-CD11br) mice using ClueGo and CluePedia plugins of Cytoscape. GO Terms (Biological Process, Cellular component, Molecular function, Immune pathway) associated with the identified proteins. The most significant parent or child term per functional group (kappa score ≥ 0.4) is shown in the functional grouped network as a group title (a). Peptides associated with the same term are represented by a node. Node color represents the class that they belong. Mixed coloring means that the specific node belongs to multiple classes. Upregulated proteins are shown in red and downregulated are shown in blue. Edges show the association of the peptide with the terms. The thickness of the edge reflects the association significance. (b) Pie chart to indicate specific GO terms (% terms per group). SI. Figure 9. Isolation and purification of microglia cells from 7-week-old WT-CD11br, vehicle or ABX-treated APPPS1-21-CD11br male and female mice (n=3). Following mechanical dissociation of cortical tissue from freshly perfused mouse brains and Percoll density centrifugation, mononuclear cells enriched for CD11b+ microglia were isolated via fluorescent activated cell sorting (a). (b) Representative flow cytometry gating strategy and co-staining of cells with CD11b-Alexa647 and CD45-Alexa488 antibodies for isolation of microglia CD11bhi-CD45lo. SI. Figure 10. Cecal analysis of APPPS1-21-CD11br transgenic mice treated with vehicle or ABX. A. (a) Representative images vehicle-treated male (M) and female (F) (top panel) and ABX-treated male (M) and female (F) (bottom panel). (b). One-way ANOVA analysis, Sidaks multiple comparison (adjusted p-val <0.0001) shows ABX-treatment resulted in significant increase in cecal weight compared to vehicle treated APPPS1-CD11br in both male and females. Heatmap to represent distinct cecal short-chain fatty acids (B), bile-acids (C) and Tryptophan (D) metabolites identified from metabolomic analysis of cecal content collected from WT-CD11br, vehicle or ABX-treated APPPS1-21-CD11br male and female mice. The metabolites with increased levels in ABX-treated mice are shown in bold. Supplemental Table 1. The list of top 10 upregulated and downregulated DEPs between vehicle-treated APPPS1-21-CD11br and WT-CD11br male and female mice. Supplemental Table 2. The list of top 10 upregulated and downregulated DEPs between ABX and vehicle-treated APPPS1-21-CD11br male and female mice. Supplemental Table 3. List of Primers used for RT-PCR and qPCR.

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