Journal: bioRxiv

Article Title: Regulation of hematopoietic stem cell (HSC) proliferation by Epithelial Growth Factor Like-7 (EGFL7)

doi: 10.1101/2025.01.21.634107

Figure Lengend Snippet: (A) 12-16 weeks old WT or Egfl7 KO mice were sacrificed, and BM cell populations were analyzed by flow cytometry, and LTC-IC assays. Total number of BM cells in Egfl7 KO mice compared to WT. (B) Number of BM LSK (Lin-Sca1+ cKit+ cells), (C) number of BM HSCs (Lin-Sca1+ cKit+ CD150+ CD48-cells) and (D) number of BM Long-Term (LT) HSCs (Lin-Sca1+ cKit+ CD150+ CD48-CD34-cells) and Short-Term (ST) HSCs (Lin-Sca1+ cKit+ CD150+ CD48-CD34-cells) in Egfl7 KO mice compared to WT. (E) Representative Z-stack immunofluorescence images of sterna from Egfl7 KO mice stained with Lin (green), CD41 (green), CD48 (green), and CD150 (red) compared to WT. (F) Quantification of number of HSCs per sternum segment in Egfl7 KO mice compared to WT. (G) Total number of LTC-IC colonies from BM of Egfl7 KO mice compared to WT. (H) Lethally irradiated WT BoyJ mice were competitively transplanted at 1:1 ratio with sort-purified CD45.2 + LSK cells from BM of Egfl7 KO or WT mice and sort-purified WT CD45.1 + LSK cells. Mice were bled every 4 weeks and peripheral blood chimerism was analyzed by flow cytometry. At 16 weeks post transplantation, mice were sacrificed, and BM cells were analyzed by flow cytometry. Percentage of CD45.2 + cells in peripheral blood of transplanted mice at 4-16 weeks post transplantation. (I) Percentage of CD45.2 + BM cells and (J) total number of BM cells in transplanted mice at 16 weeks post-transplantation. These data are representative of 3 or more experiments done with N= 4-6 mice per group. P = *<0.05, **<0.01, ***<0.001.

Article Snippet: 12-16 weeks old C57/Bl6 mice were injected i.p with 10 µg/mouse of recombinant EGFL7 protein (rEGFL7) (Peprotech) daily for 10 days.

Techniques: Flow Cytometry, Immunofluorescence, Staining, Irradiation, Purification, Transplantation Assay

Journal: bioRxiv

Article Title: Regulation of hematopoietic stem cell (HSC) proliferation by Epithelial Growth Factor Like-7 (EGFL7)

doi: 10.1101/2025.01.21.634107

Figure Lengend Snippet: (A) Lethally irradiated WT BoyJ mice were transplanted with BM from 12-16 weeks old WT or Egfl7 KO mice mixed with BM from WT BoyJ mice at 10:1 ratio. Mice were bled every 4 weeks and peripheral blood chimerism was analyzed by flow cytometry. At 16 weeks post transplantation, mice were sacrificed, and BM cells were analyzed by flow cytometry. Percentage of CD45.2 + cells in peripheral blood of transplanted mice at 4-16 weeks post-transplantation. (B) Percentage of BM CD45.2 + cells, (C) percentage of CD45.2 + BM LSK cells, and (D) percentage of CD45.2 + BM HSCs in transplanted mice at 16 weeks post-transplantation. (E) BM from above primary transplanted mice were transplanted into lethally irradiated WT BoyJ mice. Recipient secondary transplanted mice were bled every 4 weeks and peripheral blood chimerism was analyzed by flow cytometry. At 16 weeks post transplantation, secondary transplanted mice were sacrificed, and BM cells were analyzed by flow cytometry. Percentage of CD45.2 + cells in peripheral blood of secondary transplanted mice. (F) Percentage of BM CD45.2 + cells, (G) total number of BM cells, (H) percentage of CD45.2 + BM LSK cells, and (I) percentage of CD45.2 + BM HSCs in secondary transplanted mice. These data are representative of 3 or more experiments done with N= 3-6 mice per group. P = *<0.05, **<0.01, ***<0.001.

Article Snippet: 12-16 weeks old C57/Bl6 mice were injected i.p with 10 µg/mouse of recombinant EGFL7 protein (rEGFL7) (Peprotech) daily for 10 days.

Techniques: Irradiation, Flow Cytometry, Transplantation Assay

Journal: bioRxiv

Article Title: Regulation of hematopoietic stem cell (HSC) proliferation by Epithelial Growth Factor Like-7 (EGFL7)

doi: 10.1101/2025.01.21.634107

Figure Lengend Snippet: 12-16 weeks old WT or Egfl7 KO mice were injected with BrdU (50µg/gm, i.p), followed by daily BrdU treatment through drinking water (0.8 mg/ml) until mice were sacrificed at 1-, 3-, 7-, and 30-days post BrdU injection. BM populations were analyzed for BrdU incorporation by flow cytometry. (A) Percentage of HSCs in the G 0 -G 1 , S, and G 2 -M cell cycle phases in Egfl7 KO mice compared to control on day 1, (B) 3, and (C) 7 post BrdU treatment. Mice were sacrificed 30 days post BrdU treatment and BM was analyzed for BrdU incorporation by flow cytometry. (D) (left) Representative flow plots of BrdU incorporation assay at 30 days in BM HSCs from Egfl7 KO mice compared to WT. (right) Percentage of cells in G 0 -G 1 , S, and G 2 M phases in Egfl7 KO mice compared to WT. (E) BM HSCs were sorted from 12-16 weeks old WT or Egfl7 KO mice and analyzed for EGFL7 expression by immunofluorescence. Representative immunofluorescence images of BM HSCs stained for EGFL7 (red) and Ki67 (green). Nuclei are counterstained with DAPI. (F) Mean fluorescence intensity of EGFL7 in Ki67+ BM HSCs compared to Ki67-HSCs from WT mice. (G) Quantification of percentage of Ki67+ BM HSCs in Egfl7 KO mice compared to WT. These data are representative of 3 or more experiments with N=4-7 mice per group. P = *<0.05, **<0.01, ***<0.001.

Article Snippet: 12-16 weeks old C57/Bl6 mice were injected i.p with 10 µg/mouse of recombinant EGFL7 protein (rEGFL7) (Peprotech) daily for 10 days.

Techniques: Injection, BrdU Incorporation Assay, Flow Cytometry, Control, Expressing, Immunofluorescence, Staining, Fluorescence

Journal: bioRxiv

Article Title: Regulation of hematopoietic stem cell (HSC) proliferation by Epithelial Growth Factor Like-7 (EGFL7)

doi: 10.1101/2025.01.21.634107

Figure Lengend Snippet: (A) Schematic representation of experiment. WT mice were treated with rEGFL7 or PBS (control) for 10 days after which mice were sacrificed, and BM was analyzed by flow cytometry, and LTC-IC assays. (B) Total BM cells, (C) percentage of BM LSKs, (D) total BM LSKs, (E) total HSCs, (F) total BM LT-HSCs, (G) percentage of BM HSCs, and (H) percentage of BM LT-HSCs in WT mice 10 days after treatment with rEGFL7 compared to control. (I) Total number of LTC-IC colonies in BM from WT mice after treatment with rEGFL7 compared to control for 10 days. (J) BM from rEGFL7-treated or control mice was transplanted into lethally irradiated WT BoyJ mice. Mice were bled every 4 weeks and BM was analyzed at 20 weeks post-transplantation. Percentage of CD45.2 + cells in peripheral blood of transplanted mice at 4-20 weeks post-transplantation. (K) Percentage of CD45.2 + cells in BM of transplanted mice at 20 weeks post-transplantation. These data are representative of 3 or more experiments performed in triplicates with N= 3-6 mice per group. P = *<0.05, **<0.01, ***<0.001.

Article Snippet: 12-16 weeks old C57/Bl6 mice were injected i.p with 10 µg/mouse of recombinant EGFL7 protein (rEGFL7) (Peprotech) daily for 10 days.

Techniques: Control, Flow Cytometry, Irradiation, Transplantation Assay

Journal: bioRxiv

Article Title: Regulation of hematopoietic stem cell (HSC) proliferation by Epithelial Growth Factor Like-7 (EGFL7)

doi: 10.1101/2025.01.21.634107

Figure Lengend Snippet: (A) 12-16 weeks old Egfl7 KO mice were treated with rEGFL7 (Egfl7 KO – rE7) or PBS (Egfl7 KO – CNT) for 10 days after which BM was analyzed by flow cytometry. WT mice treated with PBS were used as controls (WT – CNT). Total number of BM cells, (B) total number of BM LSKs, (C) total number of BM HSCs, (D) total number of BM LT-HSCs in Egfl7 KO – rE7 and Egfl7 KO – CNT mice compared to WT – CNT. (E) 12-16 weeks old Egfl7 KO mice were treated with rEGFL7 (Egfl7 KO – rE7) or PBS (Egfl7 KO – CNT) for 10 days. For the BrdU assay, mice were injected with BrdU (50µg/gm, i.p) on day 0, followed by daily BrdU treatment through drinking water (0.8 mg/ml) until mice were sacrificed on day 11 and BM was analyzed for BrdU incorporation by flow cytometry. Percentage of BM LSK cells in G 0 -G 1 , S, and G 2 M phases in in Egfl7 KO – rE7 and Egfl7 KO – CNT mice compared to WT – CNT. (F) Schematic representation of 5-FU survival assay. 12-16 weeks old WT or Egfl7 KO mice were injected with 5-FU (150 mg/kg, i.p.) once a week for 4 weeks. Mice were monitored daily for survival. (G) Kaplan-Meier survival curve of WT and Egfl7 KO mice after 5-FU treatment. (H) 12-16 weeks old WT or Egfl7 KO mice injected with 5-FU (150 mg/kg, i.p.) were sacrificed 3 days post 5-FU injection and peripheral blood and BM was analyzed by an automated hematology analyzer. BM cells were stained with Ki67 and DAPI to analyze proliferating LSK cells, or with Annexin V to analyze apoptotic cells by flow cytometry. Number of peripheral blood WBCs in peripheral blood, (I) number of BM cells, (J) number of BM LSK cells, (K) number of BM HSCs in WT or Egfl7 KO mice on day 3 after 5-FU treatment. (L) Percentage of BM LSK cells in G 0 -G 1 , S, and G 2 M phases in WT or Egfl7 KO mice. (M) Percentage of apoptotic BM HSCs in WT or Egfl7 KO mice. These data represent 3 or more experiments performed in triplicates with N= 3-6 mice per group. P = *<0.05, **<0.01, ***<0.001.

Article Snippet: 12-16 weeks old C57/Bl6 mice were injected i.p with 10 µg/mouse of recombinant EGFL7 protein (rEGFL7) (Peprotech) daily for 10 days.

Techniques: Flow Cytometry, BrdU Staining, Injection, BrdU Incorporation Assay, Clonogenic Cell Survival Assay, Staining

Journal: bioRxiv

Article Title: Regulation of hematopoietic stem cell (HSC) proliferation by Epithelial Growth Factor Like-7 (EGFL7)

doi: 10.1101/2025.01.21.634107

Figure Lengend Snippet: (A) BM cKit + cells (HSPCs) from 12-16 weeks old WT and Egfl7 KO mice were subjected to scRNA-sequencing. Uniform Manifold Approximation and Projection (UMAP) of HSPCs from BM of Egfl7 KO and WT mice, colored by clustering and annotated based on gene signature. (B) UMAP of HSPCs colored by expression of key cell-type marker genes Hlf (HSCs), Pf4 (MPP2), Ctsg (MPP3), and Dntt (MPP4). (C) Violin plot distribution of Egfl7 expression levels across all HSPC clusters. (D) Heatmap of differentially expressed genes in HSC cluster from Egfl7 KO mice compared to WT. (E) Relative mRNA expression of Egr1 , Klf2 , Klf4 , and Mid1 in HSCs sorted from Egfl7 KO mice compared to WT, as determined by qRT-PCR. mRNA expression is calculated relative to Gapdh . N= 5 mice per group. (F) (left) Percentage of HSCs (cluster 3) in G1, S, and G2M cell cycle phases in Egfl7 KO mice compared to WT. (right) Violin plots of cell cycle scores in HSCs (cluster 3) from Egfl7 KO mice compared to WT. (G) UMAP of BM HSCs (LSK CD150+ cells) from Egfl7 KO and WT mice, colored by clustering. (H) Violin plot distribution of Hlf, (I) Mki67 and Egfl7 expression levels across all HSC clusters. (J) UMAP of HSCs colored by cell cycle scores in WT (top) and Egfl7 KO (bottom) mice. (K) Top differentially up– and down-regulated signaling pathways in Egfl7 KO HSCs compared to WT. P = *<0.05, **<0.01, ***<0.001.

Article Snippet: 12-16 weeks old C57/Bl6 mice were injected i.p with 10 µg/mouse of recombinant EGFL7 protein (rEGFL7) (Peprotech) daily for 10 days.

Techniques: Sequencing, Expressing, Marker, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Regulation of hematopoietic stem cell (HSC) proliferation by Epithelial Growth Factor Like-7 (EGFL7)

doi: 10.1101/2025.01.21.634107

Figure Lengend Snippet: (A) WT mice were treated with rEGFL7 or PBS (control) for 10 days after which BM cKit+ cells (HSPCs) were subjected to scRNA-seq. UMAP of HSPCs from BM of rEGFL7-treated and control mice, colored by clustering and annotated based on gene signature. (B) UMAP of HSPCs colored by treatment group-rEGFL7-treated (red) and control (black). (C) UMAP of HSPCs colored by expression of key cell-type marker genes Hlf (HSCs), Pf4 (MPP2), Ctsg (MPP3), and Dntt (MPP4). (D) Violin plot distribution of Hlf expression levels across all HSPC clusters. (E) Heatmap of differentially expressed genes in HSC cluster from rEGFL7-treated mice compared to control. (F) Relative mRNA expression of S100a8 , S100a9 , Vegfa , Nr4a1 , Nr4a2 , and Nfkbiz in HSCs sorted from rEGFL7-treated mice compared to control, as determined by qRT-PCR. mRNA expression is calculated relative to Gapdh . N=5 mice per group. P = *<0.05, **<0.01, ***<0.001.

Article Snippet: 12-16 weeks old C57/Bl6 mice were injected i.p with 10 µg/mouse of recombinant EGFL7 protein (rEGFL7) (Peprotech) daily for 10 days.

Techniques: Control, Expressing, Marker, Quantitative RT-PCR