Fig 1: ZEB2 Controls Tissue Identity of KCs and AMs(A) Schematic of experimental set up.(B) Expression of CD45.1 (donor) and Tim4 in total Clec4F+ KCs in Clec4f-crexZeb2fl/fl chimeras. Percentage of total chimerism of KCs in Zeb2fl/fl and Clec4f-crexZeb2fl/fl mice. Data are pooled from two experiments with n = 6–10 per group.***p < 0.001 Student’s t-test.(C) Expression of CD45.1 (donor) and CD45.2 (host) in total lung AMs in Itgax-crexZeb2fl/fl mice. Percentage of total chimerism of AMs in Zeb2fl/fl and Itgax-crexZeb2fl/fl mice. Data are pooled from two experiments with n = 5–8 per group. NS; non-significant. Student’s t-test. Percentage total chimerism calculated as ratio over the chimerism in blood Ly6Chi monocytes in the same mouse.(D and E) Heatmap of expression of cell death-associated genes per group of KCs (D) or AMs (E) from SC-RNA-seq data.(F) Representative western blots (n = 2) for RIPK3, pMLKL, and Tubulin expression by total BAL cells isolated from CD45.1 mice that were irradiated (8 Gy) and reconstituted with Rosa26-creert2xZeb2fl/fl BM. 33 weeks post reconstitution, mice were fed 5 mg tamoxifen or corn oil as a control for 5 days. 27 days after the last dose, mice were sacrificed and BAL fluid isolated. BAL fluid from 3 or 4 mice was pooled per replicate and 150,000 cells were used per lane. Ratio of band intensity was calculated using ImageJ. Data are pooled from two experiments with n = 6–8 per group. Student’s t-test was used to calculate indicated p values.(G) Venn diagram showing DE genes specific to Zeb2-/- KCs (group 3), Zeb2-/- AMs (group 3), or shared between both mac populations.(H and I) Heatmap showing expression of top core genes across KC (H) or AM (I) groups from SC-RNA-seq data. Genes in red are significantly differentially expressed. See also Figure S3.
Fig 2: ZEB2 Controls Mac Number and Surface Phenotype(A) Expression of CD64 and F4/80 by live CD45+Ly6G-Ly6C- liver cells and Clec4F and Tim4 by total liver macs in Clec4f-crexZeb2fl/fl and Zeb2fl/fl mice. Absolute number of liver macs per gram of liver and % of total macs expressing Clec4F and Tim4. Data are pooled from four experiments with n = 11–13 per group. ***p < 0.001 Student’s t test.(B) Expression of SiglecF, F4/80, and CD11b by live CD45+CD64+CD11c+ Lung macs in Itgax-crexZeb2fl/fl and Zeb2fl/fl mice. AMs as a percentage of total live CD45+ cells, absolute number, and percentage of CD11b+ and CD11b- AMs in Itgax-crexZeb2fl/fl or Zeb2fl/fl mice. Data are pooled from two experiments with n = 7–8 per group. *p < 0.05, ***p < 0.001 Student’s t test.(C and D) t-SNE plot of SC-RNA-seq data of KCs from Clec4f-crexZeb2fl/fl or Zeb2fl/fl mice (C) or AMs from Itgax-crexZeb2fl/fl or Zeb2fl/fl mice (D), showing clusters, assigned groups, and CRE- (Red) and CRE+ (Teal) overlay.(E) tSNE plots showing expression of Zeb2, Siglecf, and Ms4a1 in KCs.(F) tSNE plots showing expression of Zeb2, Epcam, and Cd101 in AMs.(G and H) Top 15 DE genes per group based on LogFC per group of KCs (G) or AMs (H). See also Figure S1.
Fig 3: Loss of LXRa from KCs Recapitulates Main Features of Zeb2-/- KCs(A) Expression of CD64 and F4/80 by live CD45+Ly6G-Ly6C- liver cells in Clec4f-crexNr1h3fl/fl and Nr1h3fl/fl mice. Liver macs as a percentage of total live CD45+ cells and absolute number per gram of liver.(B) Expression of Clec4F and Tim4 by total liver macs in Clec4f-crexNr1h3fl/fl and Nr1h3fl/fl mice and percentage of total macs expressing Clec4F and Tim4. Data are pooled from two experiments with n = 12 per group. ***p < 0.001 Student’s t test.(C) Percentage total chimerism of total Clec4F+ KCs in Nr1h3fl/fl and Clec4f-crexNr1h3fl/fl mice. Data are pooled from two experiments with n = 6–8 per group. ***p < 0.001; Student’s t-test.(D) t-SNE plot of SC-RNA-Seq data from KCs from Clec4f-crexNr1h3fl/fl and Nr1h3fl/fl mice, showing clusters, assigned groups and CRE- (Red) and CRE+ (Teal) overlay.(E) Heatmaps showing top DE genes (15 downregulated, 15 upregulated) based on LogFC in KCs with loss of LXRa and expression of the same genes by the indicated groups of KCs from Zeb2fl/fl and Clec4f-crexZeb2fl/fl mice.(F and G) Histogram and MFI of CD55 expression in (F) Zeb2+/+ (Zeb2fl/fl), SiglecF+Zeb2-/- KCs, and SiglecF-Zeb2+/- KCs from Clec4f-crexZeb2fl/fl mice and (G) Nr1h3+/+ (Nr1h3fl/fl) and Nr1h3-/- KCs from Clec4f-crexNr1h3fl/fl mice.(H) Venn diagram showing DE genes specific to Zeb2-/- KCs, Nr1h3-/- KCs, or shared between both mac populations. See also Figure S4.
Supplier Page from Thermo Fisher Scientific for TIM-4 Antibody PE