Fig 2: Isolation of brain-infiltrating leukocytes for single cell profiling of epitopes and transcriptomes(A) Overall protocol schematic.(B) Images of indicated organs without PBS perfusion (right) and with PBS perfusion (left). Scale bar: 2mm.(C) Photograph of dissociated brain at the end of enzymatic dissociation (note there are no tissue chunks present).(D) Photographs of Percoll layering sequences (1-4) and layered Percoll gradient before (lower left) and after (lower middle) centrifugation, with enlarged image pointing out the buffy leukocyte layer post-centrifugation (lower right).(E) Light microscope image of washed leukocytes post-Percoll gradient centrifugation on a hemocytometer (note the lack of debris and single cell nature of suspension). Scale bar: 100mm.(F) Bar chart showing the percent of viable cells following brain dissociation, leukocyte enrichment, and cell staining, as determined by the percent of cisplatin negative/low cells of all single cells in CyTOF experiments.See also Methods video S1.(G) Bar chart derived from CITE-seq data showing percentage of cells sequenced that were leukocytes (CD45+) from the naive and brain metastasis-burdened brain. Error bars represent SEM.(H) Stacked bar chart derived from CITE-seq data showing proportions of all identifiable cells sequenced that were leukocytes (CD45+), endothelial cells, pericytes, or metastatic cancer cells from the naive and brain metastasis-burdened brain.

Fig 3: Hepatic Immune Cell Transcriptome and Surface Proteome in MAFLDC57BL/6 mice were fed either an SD or WD for 12, 24, or 36 weeks, and livers were harvested. Total live CD45+ cells were sorted (1 mouse per time point per diet), stained with total-seq A antibodies, and loaded onto the 10X Chromium platform. After QC, 56407 cells remained.(A) UMAP showing distinct clusters among total CD45+ live cells.(B) Expression of indicated proteins based on CITE-Seq antibody binding.(C) Expression of indicated genes across the 25 clusters.(D) Annotation of the cell types within the UMAP based on both transcriptome and surface proteome.(E) Distribution of clusters from SD or WD, with SD data obtained from cells pooled after 12, 24, and 36 weeks.(F–H) Heatmaps showing top DEGs for Monocytes (F), KCs (G), and Clec4f- Macrophages (H) as assessed by comparing SD and WD samples pooled from all time points. Genes in red are conserved across multiple cell types.(I) MEM heatmap showing surface proteins whose expression was altered in at least 1 cell type during MAFLD.(J and K) CITE-Seq data were exported into FlowJo software, and (J) the KC cluster was gated and TIM4 and MerTK expression were examined at indicated time points on WD and in pooled SD-fed mice or (K) the T cell cluster was gated and CD8a and CD8ß expression were examined at 36 weeks on WD and in pooled SD-fed mice. See also Figure S2.

Fig 4: Changes in Hepatic Structural Cells in MAFLDC57BL/6 mice were fed an SD or WD for 24 or 36 weeks, and livers were harvested. Live CD45- cells were then sorted (1 mouse per time point per diet) and loaded onto the 10X Chromium platform. After QC, 33,241 cells remained.(A) UMAP showing distinct clusters among total CD45- live cells.(B) Expression of indicated genes across the 5 cell types.(C) Distribution of clusters from SD- or WD-fed mice at 24 weeks (green) or 36 weeks (red).(D) Expression of Mki67 across the different clusters.(E) Expression of Mki67 as determined by qPCR on indicated cells sorted from livers of SD- or WD-fed mice (12, 24, and 36 weeks). Data are from a single experiment, with n = 4–6 per group. *p < 0.05, **p < 0.01, Student’s t test. Error bars indicate ±SEM.(F) Heatmaps showing top 40 DEGs in the indicated cells types between SD- and WD-fed mice (24 and 36 weeks). Genes in green represent DEGs specifically altered at the 36-week time point.(G) qPCR analysis for indicated genes in indicated cell populations. Data are from a single experiment, with n = 4–5 per group. *p < 0.05, **p < 0.01. Error bars indicate ±SEM. See also Figure S4.