Fig 2: EGFR interacted with GPNMB and its expression level was downregulated after cell differentiation. (a) STRING (https://www.string-db.org/) was adopted to comprehensively analyze the data for PPI network. GPNMB interacted with EGFR. (b and c) A Co-IP assay was performed to identify if GPNMB coprecipitated with EGFR. (d and e) Relative EGFR expression was measured by qRT-PCR and Western blot assay. GAPDH was set as the internal reference. ** P < 0.01 *** P < 0.001 vs control. All results represent means ± SD of triplicate determinations. STRING: Retrieval of Interacting Genes Database; PPI: protein–protein interaction; EGFR: epidermal growth factor receptor; Co-IP: co-immunoprecipitation.

Fig 3: A population of macrophages reside around the bile duct in the healthy murine liver(A) UMAP of murine myeloid cells (71,261 cells/nuclei) isolated from Figure 1B and re-clustered with TotalVI.(B and C) Top DEGs (B) and DEPs (C) between cell types.(D) Expression of Gpnmb and Cd207.(E) Expression of VSIG4 and F4/80 (left) or MHCII, CD11c, and DAPI (right) by confocal microscopy. Capsule macs identified by white arrows. Scale bars, 50 µm.(F) Molecular Cartography of indicated genes at liver capsule.(G) Expression of VSIG4, F4/80, GLUL, and DAPI (left) or F4/80 or CCR2 (right, inset) by confocal microscopy. Scale bars, 100 µm.(H) Molecular Cartography of indicated genes at portal triad. PV, portal vein; CV, central vein; HA, hepatic artery; BD, bile duct. Arrows indicate specific cell types, where color corresponds to markers. Images are representative of 2–4 mice.(I and J) Top GO terms for KCs (I) and bile-duct LAMs (J).(K) Representative image showing expression of VSIG4 (red) CD19 (yellow) and CD3E (magenta) by MICS analysis (left) and % of B or T cells found with/without a KC per field of view (right). Data are pooled from multiple fields of view in 2 mice. ***p < 0.001 Student’s t test.(L) Mice (29-week-old) were treated with 3.5 mg/kg LPS or PBS and 2 h later, livers were harvested without the capsule. KCs and LAMs were FACS-purified and expression of Il1b, Tnf, IL10, and Il18 was examined by qPCR, compared with b-actin. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA with Bonferroni post-test. See also Figure S3 and Table S2.

Fig 4: GPNMB promoted cell viability and neuronal differentiation while shGPNMB performed differently. (a) Relative ßIII tubulin expression was measured by qRT-PCR and Western blot assay. Upregulation of ßIII tubulin expression suggested the successful differentiation of the neural stem cells into neurons. (b and c) Relative GPNMB expression was measured by qRT-PCR and Western blot assay. (d and e) Transfection efficiency was validated by qRT-PCR and Western blot assay. (f) Cell viability was measured via CCK-8. GPNMB promoted while shGPNMB inhibited cell viability. (g) Immunofluorescence assay was adopted to assess the status of neuronal differentiation (magnification 200×, scale bar 100 µm). Red part referred to the targeted proteins, and blue meant the nuclei as stained by DAPI. (h) Relative neural differentiation markers’ (ßIII tubulin and CNPase) expressions were measured by Western blot assay. GAPDH was set as the internal reference. ** P < 0.01 or *** P < 0.001 vs Control; + P < 0.05 or +++ P < 0.001 vs NC; ## P < 0.01, ### P < 0.001 vs shNC. All results represent means ± SD of triplicate determinations. qRT-PCR: quantitative reverse-transcription polymerase chain reaction; GPNMB: glycoprotein non-metastatic melanoma B; shNC: short-hairpin-negative control; CCK-8: cell counting kit-8; CNPase: 2',3'-cyclic nucleotide 3' phosphodiesterase.

Fig 5: Validated flow cytometry gating strategy for murine myeloid cells, related to Figure 2(A) CITE-seq data from the murine myeloid cells in Figure 2A were exported as an FCS file and an in silico gating strategy identified in FlowJo software.(B) Application of the in silico gating strategy with a 21-color flow cytometry panel. Myeloid cells were pre-gated as live CD45+ lineage cells (Ly6G−CD19−NK1.1−B220−CD3−). Data are representative of 3 experiments with 3–6 mice per experiment.(C) cDC1s, cDC2s, migratory cDCs (Mig. cDCs), peritoneal macs (Peri. Macs), KCs, and non-KC macs (non-KCs) were FACS-purified using gating strategy in (B), mRNA was isolated and qPCR performed to examine expression of indicated genes defining each population to validate their identity. Data are representative of 2 experiments with n = 3–6.(D) Putative peritoneal macs were FACS-purified using gating strategy in (B) and expression of Gata6 was examined by qPCR compared with other hepatic myeloid populations. Data are from a single experiment with n = 6.(E) Peritoneal macs as a % of total macs recovered from the liver using different digestion techniques (in vivo, ex vivo, or capsule) or in supernatants in which livers were washed following removal from the mouse but prior to digestion (wash). Data are from a single experiment with n = 4. ∗p < 0.05, ∗∗p < 0.01 one-way ANOVA with Bonferroni post-test compared with wash data.(F) Expression of CD14 and CD207 within the non-KC mac population from (B) (left) and % of CD207+ and CD207− populations among total macs in livers digested using the ex vivo or in vivo protocols or in dissected and digested liver capsule (right). Data are representative of two experiments with n = 4–5 mice per experiment. ∗∗∗∗p < 0.0001 mixed effects analysis with Tukey’s multiple comparison test.(G) Expression of VSIG4, F4/80, GLUL, and DAPI by confocal microscopy. Insets represent zones featured in Figures 2E, 2G, and S3I.(H) Molecular Cartography of indicated genes and cell types. Insets represent zones featured in Figures 2F, 2H, and S3J.(I) Expression of VSIG4, F4/80, GLUL, and DAPI by confocal microscopy at the central vein. Scale bars, 50 μm.(J) Molecular Cartography of indicated genes and cell types at central vein.(K) Expression of F4/80, EPCAM, CCR2, GPNMB, and DAPI by confocal microscopy at a portal vein (top) or F4/80 or GPNMB alone (bottom). Scale bars, 25 μm.(L) Quantification of % of Gpnmb & Trem2 counts over Adgre1 counts in indicated regions of tissue as assessed using Molecular Cartography data. Each dot represents an individual region. ∗p < 0.05, ∗∗∗∗p > 0.0001 one-way ANOVA with Bonferroni post-test.(M) Expression of DESMIN and F4/80 at the liver capsule and underlying parenchyma (left) or EPCAM, DESMIN and F4/80 at the bile duct by confocal microscopy. PV, portal vein; CV, central vein; HA, hepatic artery; BD, bile duct. Arrows indicate specific cell types, where color corresponds to cell type/markers. All images are representative of 2–6 mice.