Figures and data
RBP-J deficient mice display more Ly6Clo blood monocytes.
(A) Blood Ly6Chi and Ly6Clo monocytes in Rbpj+/+Lyz2cre/cre control and Rbpjfl/flLyz2cre/cre mice were determined by flow cytometry analyses (FACS). Representative FACS plots (left) and cumulative data of cell ratio (right) are shown.
(B and C) Representative FACS plots and cumulative data quantitating percentages of BM monocyte subsets (B) and myeloid progenitor cells (C) (CD45+CD11b+Ly6G- CD115+ Ly6Chi monocyte; CD45+CD11b+Ly6G-CD115+Ly6Clo monocyte; MDP, CD45+Lin-CD117+CD115+CD135+ Ly6C-; cMoP, CD45+Lin-CD11b-CD117+CD115+ CD135-Ly6C+). Lin: CD3, B220, Ter119, and Gr-1, CD11b.
(D) Experimental outline for panel (E).
(E) Cumulative data quantitating percentages of sinusoidal monocytes (CD45+) within total BM Ly6Clo monocytes.
Data are pooled from at least 2 independent experiments; n ≥ 4 in each group. Data are shown as mean ± SEM; n.s., not significant; **, P < 0.01 (two-tailed Student’s unpaired t test). Each symbol represents an individual mouse.
Monocyte subsets in RBP-J deficient mice display normal cell death.
(A) Representative FACS plots of monocyte subsets in blood stained with 7-AAD and Annexin V.
(B) FACS analysis of Ki-67 expression in Rbpj+/+Lyz2cre/cre control and Rbpjfl/flLyz2cre/cre blood monocyte subsets. Black lines represent control mice and red lines represent RBP-J deficient mice.
(C) Analysis of time course of EdU incorporation of monocyte subsets in BM and blood after a single 1 mg EdU pulsing. The percentages of EdU+ cells among the indicated monocyte subsets are shown.
(D) Experimental outline for panel (E).
(E) Analysis of time course of latex beads incorporation of monocyte subsets in blood after latex beads injection. The percentages of latex+ cells among the indicated monocyte subsets are shown.
(F) Cartoon depicting the adoptive transfer. BM GFP+Ly6Chi monocytes were sorted from Lyz2cre/creCx3cr1gfp/+or Rbpjfl/flLyz2cre/creCx3cr1gfp/+mice and transferred into Ccr2RFP/RFP recipient mice. Sixty hours after transfer, cell fate was analyzed.
(G) Representative FACS plots are shown in the left panel, and the frequencies of GFP+Ly6Clo monocytes within total Ly6Clo monocytes are shown in the right panel.
Data are pooled from 2 independent experiments (G); n ≥ 2 in each group (C, E and G). Data are shown as mean ± SEM; n.s., not significant; (two-tailed Student’s unpaired t test). Each symbol represents an individual mouse (E and G).
The role of RBP-J in blood Ly6Clo monocytes is cell intrinsic.
(A) Cartoon depicting the bone marrow transplantation. BM recipient 6-week-old male C57BL/6 mice (CD45.2) were lethally irradiated. BM cells from donor mice (Rbpj+/+Lyz2cre/cre or Rbpjfl/flLyz2cre/cre and CD45.1, Cx3cr1gfp/+) were collected and transferred into recipient mice. Mice were used after 8 weeks of bone marrow reconstitution.
(B) Representative FACS plots (left) and cumulative data (right) quantitating the frequency of Rbpj+/+Lyz2cre/cre and Rbpjfl/flLyz2cre/cre donor cells among Ly6Chi and Ly6Clo monocytes in the blood of recipient mice.
(C) Cartoon depicting the generation of Rbpj+/+Lyz2cre/cre control or Rbpjfl/flLyz2cre/cre and Cx3cr1gfp/+ parabiotic pairs.
(D) Representative FACS plots (left) and cumulative data (right) quantitating percentages of monocyte subsets derived from control or RBP-J deficient mice in control or RBP-J deficient mice.
(E) Representative FACS plots (left) and cumulative data (right) quantitating percentages of monocyte subsets derived from control or RBP-J deficient mice in Cx3cr1gfp/+ mice.
Data are pooled from at least 2 independent experiments; n ≥ 6 in each group. Data are shown as mean ± SEM; n.s., not significant; ***, P < 0.001; ****, P < 0.0001 (two– tailed Student’s unpaired t test). Each symbol represents an individual mouse. SSC-A, side scatter area.
Phenotypic markers in Ly6Clo monocytes are changed in RBP-J deficient mice.
(A) Heatmap of RNA-seq dataset showing the top 20 downregulated and upregulated genes in blood Ly6Clo monocytes from Rbpjfl/flLyz2cre/cre versus Rbpj+/+Lyz2cre/cre control mice. Blue and red font indicates downregulated and upregulated genes in RBP-J deficient Ly6Clo monocytes respectively.
(B) Representative FACS plots, cumulative MFI, and qPCR analysis of CCR2/CD11c expression in control and RBP-J deficient blood monocyte subsets. Shaded curves represent isotype control, black lines represent control mice and red lines represent RBP-J deficient mice.
(C) PCA of indicated cell types.
(D) Representative FACS plots and cumulative MFI of CCR2/CD11c expression in blood monocyte subsets derived from control or RBP-J deficient mice. Black lines represent control mice, and red lines represent RBP-J deficient mice.
Data are pooled from 2 independent experiments (B and D); n ≥ 6 in each group. Data are shown as mean ± SEM; n.s., not significant; **, P < 0.01; ****, P < 0.0001 (two– tailed Student’s unpaired t test). Each symbol represents an individual mouse.
Blood Ly6Clo monocytes are decreased in DKO mice.
(A) Representative FACS plots and cumulative MFI of CCR2 expression in Lyz2cre/creCcr2RFP/+ control, Rbpjfl/flLyz2cre/creCcr2RFP/+ and Rbpjfl/flLyz2cre/creCcr2RFP/RFP (DKO) blood Ly6Chi and Ly6Clo monocytes are shown.
(B and C) Blood monocyte subsets in control, RBP-J deficient and DKO mice were determined by FACS. Representative FACS plots (B) and cumulative data of cell ratio
(C) are shown.
(D) Representative FACS plots and cumulative MFI of CD11c expression in control, RBP-J deficient and DKO blood Ly6Chi and Ly6Clo monocytes are shown.
Data are pooled from at least 2 independent experiments; n ≥ 5 in each group. Data are shown as mean ± SEM; n.s., not significant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (two-tailed Student’s unpaired t test). Each symbol represents an individual mouse.
RBP-J deficient mice exhibit more lung Ly6Clo monocytes and CD16.2+ IM.
(A and B) Indicated populations in the lungs of Rbpj+/+Lyz2cre/cre and Rbpjfl/flLyz2cre/cre mice were determined by FACS. Representative FACS plots (A) and cumulative data of cell ratio and absolute numbers (B) are shown.
(C) Immunofluorescence staining for GFP+ cells in the lungs from Lyz2cre/creCx3cr1gfp/+and Rbpjfl/flLyz2cre/creCx3cr1gfp/+mice (CX3CR1 [green]; DAPI [blue]). Scale bars represent 50 µm.
(D and E) Rbpj+/+Lyz2cre/cre and Rbpjfl/flLyz2cre/cre mice were instilled intranasally with PBS or PBS containing LPS, and lungs were harvested at the indicated time points. Representative FACS plots (D) and cumulative data of cell ratio and absolute numbers
(E) are shown.
Data are pooled from at least 2 independent experiments; n ≥ 4 in each group. Data are shown as mean ± SEM; n.s., not significant; *, P < 0.05; **, P < 0.01; ****, P < 0.0001 (two-tailed Student’s unpaired t test). Each symbol represents an individual mouse.
DKO mice lack lung Ly6Clo monocytes and CD16.2+ IM.
(A) Representative FACS plots of lung monocyte and IM subsets in Lyz2cre/creCcr2RFP/+, Rbpjfl/flLyz2cre/creCcr2RFP/+, Lyz2cre/creCcr2RFP/RFP and Rbpjfl/flLyz2cre/creCcr2RFP/RFP mice.
(B and C) Cumulative data of cell ratio and absolute numbers of monocyte (B) and IM
(C) subsets.
Data are pooled from 2 independent experiments; n ≥ 4 in each group. Data are shown as mean ± SEM; n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (two-tailed Student’s unpaired t test). Each symbol represents an individual mouse.
Primers sequences for regular qPCR used in this study
Control and RBP-J deficient mice show similar neutrophils.
(A) qPCR analysis of Rbpj expression in sorted monocyte subsets from control and RBP-J deficient mice.
(B) Blood and BM CD45+CD11b+Ly6G+ neutrophils were determined by FACS. Cumulative data of cell ratio are shown.
(C) Spleen monocyte subsets were determined by FACS. Representative FACS plots (left) and cumulative data of cell ratio and absolute numbers (right) are shown.
Data are pooled from at least 2 independent experiments; n ≥ 2 in each group. Data are shown as mean ± SEM; n.s., not significant; *, P < 0.05 (two-tailed Student’s unpaired t test). Each symbol represents an individual mouse.
The conversion of Ly6Chi monocyte is identical in control and RBP-J deficient mice.
(A) Representative FACS plots, cumulative MFI and qPCR analysis of Nr4a1 expression in control and RBP-J deficient blood monocyte subsets. Shaded curves represent isotype control, black lines represent control mice, and red lines represent RBP-J deficient mice.
(B) Representative FACS plots of GFP+Ly6Chi monocytes in recipient.
Data are pooled from 2 independent experiments (A); n ≥ 4 in each group. Data are shown as mean ± SEM; n.s., not significant (two-tailed Student’s unpaired t test). Each symbol represents an individual mouse.
Cell-intrinsic requirement of RBP-J for Ly6Clo maintenance.
(A) Representative FACS plots (left) and cumulative data (right) quantitating percentages of blood neutrophils in recipient mice.
(B) BM neutrophils and Ly6Chi monocytes in recipient mice were determined by FACS.
(C) CD4+ T cells and CD19+ B cells were analyzed by FACS in parabiotic mice.
Data are pooled from at least 2 independent experiments (A and C); n ≥ 4 in each group. Data are shown as mean ± SEM; n.s., not significant (two-tailed Student’s unpaired t test). Each symbol represents an individual mouse.
Normal expression of CCR2 and CD11c in BM monocytes.
(A) Cumulative MFI of CCR2 expression in BM monocyte subsets.
(B) FACS plots of CCR2 and CD11c in BM Ly6Chi and Ly6Clo monocytes from control and RBP-J deficient mice.
Data are pooled from 3 independent experiments (A); n ≥ 7 in each group. Data are shown as mean ± SEM; n.s., not significant (two-tailed Student’s unpaired t test). Each symbol represents an individual mouse.
RBP-J is not required for turnover of lung Ly6Clo monocytes and CD16.2+ IM.
(A and B) Incorporation of EdU was assessed 24 hours after injection. BM monocyte subsets were used as controls. FACS plots (A) and representative data (B) are shown. Each symbol represents an individual mouse.
