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rbpj  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rbpj
    miR-UL36 impairs Notch signaling through targeting Notch3 and <t>RBPJ.</t> ( A ) HEK293 cells were transfected with a 4× CSL-Luciferase construct as well as a plasmid expressing the intracellular domain of Notch3 and either negative control miRNA mimic, miR-US33, miR-UL36, or miR-UL112. Protein lysates were harvested 24 h post-transfection, and luciferase expression was monitored using a luminometer ( n = 3; P < 0.05 calculated using a one-sample t and Wilcoxon test). ( B and C ) NHDFs were transfected with negative control miRNA mimic, miR-UL36, or siRNA against Notch3 ( B ) or RBPJ ( C ). Protein lysates were harvested 48 h post-transfection, and immunoblots were performed using the <t>indicated</t> <t>antibodies.</t> Densitometry was performed using ImageJ software for three independent experiments and plotted in the right-hand panels. ( D and E ). NHDF were mock-infected or infected with WT HCMV or a miR-UL36 mutant virus for 48 h. Protein lysates were harvested, and immunoblotting was performed using the indicated antibodies. Densitometry was performed using ImageJ software for three independent experiments and plotted in the right-hand panels. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 as determined by two-tailed t -test. ( F ) RNA was harvested from cells infected as in ( D and E ) and subjected to qRT-PCR using primers for the Notch target genes HES1 and HEY1. ( n = 3; * P < 0.05 by two-tailed t -test).
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    Images

    1) Product Images from "HCMV promotes viral reactivation through the coordinated regulation of Notch signaling by UL8 and miR-UL36"

    Article Title: HCMV promotes viral reactivation through the coordinated regulation of Notch signaling by UL8 and miR-UL36

    Journal: mBio

    doi: 10.1128/mbio.03377-25

    miR-UL36 impairs Notch signaling through targeting Notch3 and RBPJ. ( A ) HEK293 cells were transfected with a 4× CSL-Luciferase construct as well as a plasmid expressing the intracellular domain of Notch3 and either negative control miRNA mimic, miR-US33, miR-UL36, or miR-UL112. Protein lysates were harvested 24 h post-transfection, and luciferase expression was monitored using a luminometer ( n = 3; P < 0.05 calculated using a one-sample t and Wilcoxon test). ( B and C ) NHDFs were transfected with negative control miRNA mimic, miR-UL36, or siRNA against Notch3 ( B ) or RBPJ ( C ). Protein lysates were harvested 48 h post-transfection, and immunoblots were performed using the indicated antibodies. Densitometry was performed using ImageJ software for three independent experiments and plotted in the right-hand panels. ( D and E ). NHDF were mock-infected or infected with WT HCMV or a miR-UL36 mutant virus for 48 h. Protein lysates were harvested, and immunoblotting was performed using the indicated antibodies. Densitometry was performed using ImageJ software for three independent experiments and plotted in the right-hand panels. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 as determined by two-tailed t -test. ( F ) RNA was harvested from cells infected as in ( D and E ) and subjected to qRT-PCR using primers for the Notch target genes HES1 and HEY1. ( n = 3; * P < 0.05 by two-tailed t -test).
    Figure Legend Snippet: miR-UL36 impairs Notch signaling through targeting Notch3 and RBPJ. ( A ) HEK293 cells were transfected with a 4× CSL-Luciferase construct as well as a plasmid expressing the intracellular domain of Notch3 and either negative control miRNA mimic, miR-US33, miR-UL36, or miR-UL112. Protein lysates were harvested 24 h post-transfection, and luciferase expression was monitored using a luminometer ( n = 3; P < 0.05 calculated using a one-sample t and Wilcoxon test). ( B and C ) NHDFs were transfected with negative control miRNA mimic, miR-UL36, or siRNA against Notch3 ( B ) or RBPJ ( C ). Protein lysates were harvested 48 h post-transfection, and immunoblots were performed using the indicated antibodies. Densitometry was performed using ImageJ software for three independent experiments and plotted in the right-hand panels. ( D and E ). NHDF were mock-infected or infected with WT HCMV or a miR-UL36 mutant virus for 48 h. Protein lysates were harvested, and immunoblotting was performed using the indicated antibodies. Densitometry was performed using ImageJ software for three independent experiments and plotted in the right-hand panels. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 as determined by two-tailed t -test. ( F ) RNA was harvested from cells infected as in ( D and E ) and subjected to qRT-PCR using primers for the Notch target genes HES1 and HEY1. ( n = 3; * P < 0.05 by two-tailed t -test).

    Techniques Used: Transfection, Luciferase, Construct, Plasmid Preparation, Expressing, Negative Control, Western Blot, Software, Infection, Mutagenesis, Virus, Two Tailed Test, Quantitative RT-PCR



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    Identification of duck embryo fibroblasts and the effects of <t>RBPJ</t> overexpression on proliferation. (A) Immunofluorescence identification of decorin and vimentin in duck embryo fibroblasts. Target proteins (red, Alexa Fluor 488), nuclei (blue, Hochest 33342). Scale bar =100 μm. (B) Relative expression level of RBPJ mRNA, plot with Control as 1. <t>(C)</t> <t>GAPDH</t> and RBPJ protein band diagrams. (D) Quantified relative protein bands, plot with Control as 1. (E) Cell viability curve after RBPJ overexpression. (F) Staining maps of Group C and OE. (G) Quantitative plot of the proportion of EdU positive cells in two groups. pCD3.1 is the plasmid of group C and pCD3.1-RBPJ is the plasmid of group OE of RBPJ ( ⁎⁎ P < 0.01; ⁎⁎⁎ P < 0.001; ⁎⁎⁎⁎ P < 0.0001).
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    (a) Schematic illustrating the experimental design for labeling proliferating MG. (b) Schematic illustrating the changes in MG proliferation over time. (c) Representative EdU immunostaining on retinal sections from Glast-Cre ERT2 ;Sun1:GFP mice treated with CCA at P28 and collected 4 months post-treatment. (d) Schematic illustration of scRNA-seq experiment. (e) UMAP plot of scRNA-seq data for MG treated with CCA and control virus. (f) Split UMAP plots of the control and CCA groups. (g) Feature plots of normalized Notch related genes, Notch1 , <t>Rbpj</t> and Hes1 gene expression in different cell clusters.
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    miR-UL36 impairs Notch signaling through targeting Notch3 and <t>RBPJ.</t> ( A ) HEK293 cells were transfected with a 4× CSL-Luciferase construct as well as a plasmid expressing the intracellular domain of Notch3 and either negative control miRNA mimic, miR-US33, miR-UL36, or miR-UL112. Protein lysates were harvested 24 h post-transfection, and luciferase expression was monitored using a luminometer ( n = 3; P < 0.05 calculated using a one-sample t and Wilcoxon test). ( B and C ) NHDFs were transfected with negative control miRNA mimic, miR-UL36, or siRNA against Notch3 ( B ) or RBPJ ( C ). Protein lysates were harvested 48 h post-transfection, and immunoblots were performed using the <t>indicated</t> <t>antibodies.</t> Densitometry was performed using ImageJ software for three independent experiments and plotted in the right-hand panels. ( D and E ). NHDF were mock-infected or infected with WT HCMV or a miR-UL36 mutant virus for 48 h. Protein lysates were harvested, and immunoblotting was performed using the indicated antibodies. Densitometry was performed using ImageJ software for three independent experiments and plotted in the right-hand panels. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 as determined by two-tailed t -test. ( F ) RNA was harvested from cells infected as in ( D and E ) and subjected to qRT-PCR using primers for the Notch target genes HES1 and HEY1. ( n = 3; * P < 0.05 by two-tailed t -test).
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    Nitrate functions by Notch pathway inhibition positively transcripting TFF2 in vitro. (A) Diagram of the wild‐type and mutant sequences for the three predicted <t>RBPJ</t> binding sites within the 251 bp TFF2 promoter probes. (B) Representative electrophoretic mobility shift assay (EMSA) autoradiograph. Hot probe is the biotin‐labeled wild‐type oligonucleotides of the truncated TFF2 promoter containing the binding motif; Mutant probe is the labeled oligonucleotides sequence with nucleotides mutated. The cold probe is nonlabeled competitive wild‐type probes (100 and 50 that of the concentrations). The shifted bands are indicated by arrows, which suggested the formation of DNA–protein complexes (lane 2, 3, 7). The super shifted bands indicated the formation of DNA–protein–antibody complexes (lane 3, 7). “+” and “−” represent presence and absence, respectively. (C) Relative TFF2 promoter (Full, Mut1, Mut2, and Mut3) luciferase activity was detected by DLR assays in RBPJ overexpressed and <t>normal‐expressed</t> <t>GES‐1</t> cells. (D) A schematic diagram showing the location of RBPJ putative binding regions on the TFF2 promoter. (E) RT‐qPCR analysis of TFF2 binding site expression of GES‐1 cells. Target site expression in RBPJ‐treated groups was normalized to IgG negative control groups and expressed as fold change relative to the IgG groups. (F) IF staining of NICD (pink) and DAPI (blue). Scale bar = 50 µm. (G) IF analysis of MFI of nuclear NICD in positive cells. (H) Representative immunoblotting band of Notch signaling pathway in GES‐1 cells. (I–K) Analyses of immunoblotting band gray value of (H). (L) RT‐qPCR analysis of TFF2 mRNA expression of DMSO/DAPT treated GES‐1 cells. Target gene expression was normalized to GAPDH mRNA and expressed as fold change relative to the DMSO vehicle group. (M) RT‐qPCR analysis of TFF2 mRNA expression in RBPJ overexpressed and NICD deprived GES‐1 cells. The target gene expression was normalized to GAPDH mRNA and expressed as fold change relative to the vector + DMSO group. (N) RT‐qPCR analysis of TFF2 mRNA expression in RBPJ overexpressed and NICD‐RBPJ overexpressed GES‐1 cells. The target gene expression was normalized to GAPDH mRNA and expressed as fold change relative to the vector1 + vector2 group. (O) Representative image of PLA of NICD–RBPJ proximity. Each red dot represents a positive signal of NICD–RBPJ interaction and nuclei were counterstained with DAPI (blue). Scale bar = 20 µm. Quantitative data are expressed as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and ns denotes no significance. Nit, nitrate; EtOH, ethanol; GES‐1, human gastric epithelial; NICD, intracellular structural domain; RBPJ, recombination signal binding protein for immunoglobulin kappa J region; EMSA, electrophoretic mobility shift assay; TFF2, trefoil factor 2; DAPI, 2‐(4‐amidinophenyl)‐6‐indolecarbamidine dihydrochloride; Yhhu‐3792, N2‐(4‐isopropylphenyl)‐5‐(3‐methoxyphenoxy) quinazoline‐2,4‐diamine; Luc, luciferase; SD, standard deviation; DLR, dual‐luciferase report; PLA, proximity ligation assay.
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    (A) Bar plots showing the frequency of LSK (left) and HSCs (LSK CD48 − CD150 + ) (right) in <t>RBPj</t> vav1 WT and KO E14.5 FL embryos. Each dot represents one embryo (n=10). (B) Box plots showing cell cycle analysis of RBPj vav1 WT and KO HSCs (LSK CD48 − CD150 + ) from E14.5 FL, assessed by Ki67 and DAPI staining. Each dot represents one embryo (n=10). (C) Competitive transplantation of the indicated numbers of E14.5 FL cells from RBPj vav1 WT and KO mice (CD45.2 + ) together with 2×10 5 <t>C57BL/6</t> BM competitor cells (CD45.1 + ) into lethally irradiated recipient mice (CD45.1 + ). Graph shows the percentage of engraftment in BM 16 weeks after primary transplantation. Each dot represents one mouse (n=4-8). (D) Limiting dilution assay (LDA) to determine the colony-repopulating unit (CRU) frequency of E14.5 FL HSCs from RBPj vav1 WT and KO embryos, analyzed by ELDA (Extreme Limiting Dilution Analysis) at 16 weeks post-transplantation. The table summarizes the estimated HSC frequencies obtained from the LDA. N=4-8 mice. (E) UMAP representation of active, intermediate and dormant HSC populations from RBPj vav1 WT and KO E14.5 FL LSK scRNA-seq data, based on the Dormant HSC signature and the Active_HSC_MPPs signature. N=2 pooled embryos. (F) Expression of HSC-related genes (upper; Hlf, Mecom, Procr, Mllt3) and active HSC-associated genes (bottom; CD34, Myc, Cdk4, Cdk6) in HSC subcluster represented in UMAP. N=2 pooled embryos. (G-I) Box plots showing score representation of HSC Score (G); Signature scores including adult HSC dormancy, serial-engrafting FL HSCs, diapause, low-output, activated HSC/MPPs, high-output, MSigDB, Hallmark Myc target genes version 1 (V1) and version 2 (V2) (H); and Dormancy module score (I) in dormant HSC populations from RBPj vav1 WT and KO E14.5 FL LSK scRNA-seq data. N=2 pooled embryos. (J) Box plots showing cell cycle analysis of WT and NICD vav1 HSCs (LSK CD48 − CD150 + ) from E14.5 FL. Each dot represents one embryo (n=6). (K) Bar plots showing the frequency of LSK (left), HSCs (LSK CD48 − CD150 + ) (middle) and MPP3/MPP4 (LSK CD48 + CD150 − ) in WT and NICD vav1 E14.5 FL. Each dot represents one embryo (n=6). (L) Bar plot showing GSEA results from bulk RNA-seq analysis comparing NICD vav1 versus WT HSCs (LSK CD48 − CD150 + ), using gene signatures previously associated with dormant HSCs, activated HSCs/MPPs, metabolic activity, cell cycle and Notch signaling, including the Notch_signaling hallmark (MSigDB_HALLMARK) as well as signatures for High-output, serially engrafting FL HSCs and serially engrafting BM HSCs. Only statistically significant pathways are shown (adjusted p-value < 0.1). Pathways are sorted in descending order of normalized enrichment score (NES). The experiment included five samples in total (n=3 NICD vav1 HSCs and n=2 WT HSCs). Box plots: center line indicates the median; lower and upper hinges represent the first and third quartiles; whiskers extend to the largest and smallest values within 1.5× the interquartile range (IQR). No outliers are present.
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    anti rbpj  (Cell Signaling Technology Inc)
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    (A) Bar plots showing the frequency of LSK (left) and HSCs (LSK CD48 − CD150 + ) (right) in <t>RBPj</t> vav1 WT and KO E14.5 FL embryos. Each dot represents one embryo (n=10). (B) Box plots showing cell cycle analysis of RBPj vav1 WT and KO HSCs (LSK CD48 − CD150 + ) from E14.5 FL, assessed by Ki67 and DAPI staining. Each dot represents one embryo (n=10). (C) Competitive transplantation of the indicated numbers of E14.5 FL cells from RBPj vav1 WT and KO mice (CD45.2 + ) together with 2×10 5 <t>C57BL/6</t> BM competitor cells (CD45.1 + ) into lethally irradiated recipient mice (CD45.1 + ). Graph shows the percentage of engraftment in BM 16 weeks after primary transplantation. Each dot represents one mouse (n=4-8). (D) Limiting dilution assay (LDA) to determine the colony-repopulating unit (CRU) frequency of E14.5 FL HSCs from RBPj vav1 WT and KO embryos, analyzed by ELDA (Extreme Limiting Dilution Analysis) at 16 weeks post-transplantation. The table summarizes the estimated HSC frequencies obtained from the LDA. N=4-8 mice. (E) UMAP representation of active, intermediate and dormant HSC populations from RBPj vav1 WT and KO E14.5 FL LSK scRNA-seq data, based on the Dormant HSC signature and the Active_HSC_MPPs signature. N=2 pooled embryos. (F) Expression of HSC-related genes (upper; Hlf, Mecom, Procr, Mllt3) and active HSC-associated genes (bottom; CD34, Myc, Cdk4, Cdk6) in HSC subcluster represented in UMAP. N=2 pooled embryos. (G-I) Box plots showing score representation of HSC Score (G); Signature scores including adult HSC dormancy, serial-engrafting FL HSCs, diapause, low-output, activated HSC/MPPs, high-output, MSigDB, Hallmark Myc target genes version 1 (V1) and version 2 (V2) (H); and Dormancy module score (I) in dormant HSC populations from RBPj vav1 WT and KO E14.5 FL LSK scRNA-seq data. N=2 pooled embryos. (J) Box plots showing cell cycle analysis of WT and NICD vav1 HSCs (LSK CD48 − CD150 + ) from E14.5 FL. Each dot represents one embryo (n=6). (K) Bar plots showing the frequency of LSK (left), HSCs (LSK CD48 − CD150 + ) (middle) and MPP3/MPP4 (LSK CD48 + CD150 − ) in WT and NICD vav1 E14.5 FL. Each dot represents one embryo (n=6). (L) Bar plot showing GSEA results from bulk RNA-seq analysis comparing NICD vav1 versus WT HSCs (LSK CD48 − CD150 + ), using gene signatures previously associated with dormant HSCs, activated HSCs/MPPs, metabolic activity, cell cycle and Notch signaling, including the Notch_signaling hallmark (MSigDB_HALLMARK) as well as signatures for High-output, serially engrafting FL HSCs and serially engrafting BM HSCs. Only statistically significant pathways are shown (adjusted p-value < 0.1). Pathways are sorted in descending order of normalized enrichment score (NES). The experiment included five samples in total (n=3 NICD vav1 HSCs and n=2 WT HSCs). Box plots: center line indicates the median; lower and upper hinges represent the first and third quartiles; whiskers extend to the largest and smallest values within 1.5× the interquartile range (IQR). No outliers are present.
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    Image Search Results


    Identification of duck embryo fibroblasts and the effects of RBPJ overexpression on proliferation. (A) Immunofluorescence identification of decorin and vimentin in duck embryo fibroblasts. Target proteins (red, Alexa Fluor 488), nuclei (blue, Hochest 33342). Scale bar =100 μm. (B) Relative expression level of RBPJ mRNA, plot with Control as 1. (C) GAPDH and RBPJ protein band diagrams. (D) Quantified relative protein bands, plot with Control as 1. (E) Cell viability curve after RBPJ overexpression. (F) Staining maps of Group C and OE. (G) Quantitative plot of the proportion of EdU positive cells in two groups. pCD3.1 is the plasmid of group C and pCD3.1-RBPJ is the plasmid of group OE of RBPJ ( ⁎⁎ P < 0.01; ⁎⁎⁎ P < 0.001; ⁎⁎⁎⁎ P < 0.0001).

    Journal: Poultry Science

    Article Title: Multi-omics analysis reveals RBPJ-mediated regulation of EGF/ACTN2 / MYPN / COL21A1 in fibroblast during oviduct functional remodeling of duck

    doi: 10.1016/j.psj.2026.106688

    Figure Lengend Snippet: Identification of duck embryo fibroblasts and the effects of RBPJ overexpression on proliferation. (A) Immunofluorescence identification of decorin and vimentin in duck embryo fibroblasts. Target proteins (red, Alexa Fluor 488), nuclei (blue, Hochest 33342). Scale bar =100 μm. (B) Relative expression level of RBPJ mRNA, plot with Control as 1. (C) GAPDH and RBPJ protein band diagrams. (D) Quantified relative protein bands, plot with Control as 1. (E) Cell viability curve after RBPJ overexpression. (F) Staining maps of Group C and OE. (G) Quantitative plot of the proportion of EdU positive cells in two groups. pCD3.1 is the plasmid of group C and pCD3.1-RBPJ is the plasmid of group OE of RBPJ ( ⁎⁎ P < 0.01; ⁎⁎⁎ P < 0.001; ⁎⁎⁎⁎ P < 0.0001).

    Article Snippet: The membrane was then incubated overnight at 4°C with the following primary antibodies diluted in Primary Antibody Dilution Buffer (P0023A, Beyotime, China): RBPJ (mouse monoclonal antibody, 1:2000, 66132-1-Ig, Proteintech, China), glyceraldehyde-3-phosphate dehydrogenase ( GAPDH , rabbit polyclonal antibody, 1:1000, 10494-1-AP, Proteintech, China) and β-tubulin (mouse monoclonal antibody,1:10000, 66240-1-Ig, Proteintech, China) (GAPDH and β-tubulin was used as the internal reference gene for normalization).

    Techniques: Over Expression, Immunofluorescence, Expressing, Control, Staining, Plasmid Preparation

    (a) Schematic illustrating the experimental design for labeling proliferating MG. (b) Schematic illustrating the changes in MG proliferation over time. (c) Representative EdU immunostaining on retinal sections from Glast-Cre ERT2 ;Sun1:GFP mice treated with CCA at P28 and collected 4 months post-treatment. (d) Schematic illustration of scRNA-seq experiment. (e) UMAP plot of scRNA-seq data for MG treated with CCA and control virus. (f) Split UMAP plots of the control and CCA groups. (g) Feature plots of normalized Notch related genes, Notch1 , Rbpj and Hes1 gene expression in different cell clusters.

    Journal: bioRxiv

    Article Title: Synergistic Inhibition of Notch Signaling and Forced Cell Cycle Re-entry Drive Müller Glia Reprogramming in Uninjured Mouse Retina

    doi: 10.64898/2026.03.09.710684

    Figure Lengend Snippet: (a) Schematic illustrating the experimental design for labeling proliferating MG. (b) Schematic illustrating the changes in MG proliferation over time. (c) Representative EdU immunostaining on retinal sections from Glast-Cre ERT2 ;Sun1:GFP mice treated with CCA at P28 and collected 4 months post-treatment. (d) Schematic illustration of scRNA-seq experiment. (e) UMAP plot of scRNA-seq data for MG treated with CCA and control virus. (f) Split UMAP plots of the control and CCA groups. (g) Feature plots of normalized Notch related genes, Notch1 , Rbpj and Hes1 gene expression in different cell clusters.

    Article Snippet: Rbpj flox/flox mice (strain: Rbpj em2Lutzy ), tdTomato reporter mice (strain B6.Cg-Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze /J ) , Sun1:GFP reporter mice (Strain B6.129-Gt(ROSA)26Sor tm5.1(CAG-Sun1/sfGFP)Nat /MmbeJ ) , and Glast-Cre ERT2 reporter mice (strain Tg(Slc1a3-cre/ERT)1Nat/J )( ) mice were purchased from the Jackson Laboratory.

    Techniques: Labeling, Immunostaining, Control, Virus, Gene Expression

    (a) Schematic of Glast-Cre ERT2 ;Rbpj flox/flox ;tdT mouse used in this study. (b) Schematic illustration of the examination of Notch inhibition via Rbpj deletion. (c) Hes1 mRNA in situ hybridization in Glast-Cre ERT2 ;tdT (Ctrl), Glast-Cre ERT2 ;Rbpj flox/wt ;tdT and Glast-Cre ERT2 ;Rbpj flox/flox ;tdT mice received tamoxifen (TAM) injection. (d) Magnified views of the highlighted regions in (c). (e) The average pixel level of Hes1 mRNA per GFP+ cell. n≥3 mice, data are presented as mean ± SEM. ns=not significant, **** P < 0.0001, by one-way ANOVA with Tukey’s post hoc test.

    Journal: bioRxiv

    Article Title: Synergistic Inhibition of Notch Signaling and Forced Cell Cycle Re-entry Drive Müller Glia Reprogramming in Uninjured Mouse Retina

    doi: 10.64898/2026.03.09.710684

    Figure Lengend Snippet: (a) Schematic of Glast-Cre ERT2 ;Rbpj flox/flox ;tdT mouse used in this study. (b) Schematic illustration of the examination of Notch inhibition via Rbpj deletion. (c) Hes1 mRNA in situ hybridization in Glast-Cre ERT2 ;tdT (Ctrl), Glast-Cre ERT2 ;Rbpj flox/wt ;tdT and Glast-Cre ERT2 ;Rbpj flox/flox ;tdT mice received tamoxifen (TAM) injection. (d) Magnified views of the highlighted regions in (c). (e) The average pixel level of Hes1 mRNA per GFP+ cell. n≥3 mice, data are presented as mean ± SEM. ns=not significant, **** P < 0.0001, by one-way ANOVA with Tukey’s post hoc test.

    Article Snippet: Rbpj flox/flox mice (strain: Rbpj em2Lutzy ), tdTomato reporter mice (strain B6.Cg-Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze /J ) , Sun1:GFP reporter mice (Strain B6.129-Gt(ROSA)26Sor tm5.1(CAG-Sun1/sfGFP)Nat /MmbeJ ) , and Glast-Cre ERT2 reporter mice (strain Tg(Slc1a3-cre/ERT)1Nat/J )( ) mice were purchased from the Jackson Laboratory.

    Techniques: Inhibition, In Situ Hybridization, Injection

    (a) Schematic of Glast-Cre ERT2 ;Rbpj flox/flox ;Sun1:GFP mouse used in this study. (b) Schematic illustration of the experiment examining how Rbpj deletion affects neurogenesis in retinal progenitor cells. Glast-Cre ERT2 ;tdT (Ctrl), Glast-Cre ERT2 ;Rbpj flox/wt ;tdT and Glast-Cre ERT2 ;Rbpj flox/flox ;tdT mice were received TAM injection at postnatal day 1 (P1) and harvested at P12. (c) Representative immunostaining of Otx2 and Sox9 on retinal. (d) Percentage of tdT+ rod photoreceptor cells in overall tdT+ cells. (e) Percentage of tdT+ Sox9+ cells in overall tdT+ cells. n≥3 mice, ns. not significant, **** P < 0.0001, by one-way ANOVA with Tukey post hoc test.

    Journal: bioRxiv

    Article Title: Synergistic Inhibition of Notch Signaling and Forced Cell Cycle Re-entry Drive Müller Glia Reprogramming in Uninjured Mouse Retina

    doi: 10.64898/2026.03.09.710684

    Figure Lengend Snippet: (a) Schematic of Glast-Cre ERT2 ;Rbpj flox/flox ;Sun1:GFP mouse used in this study. (b) Schematic illustration of the experiment examining how Rbpj deletion affects neurogenesis in retinal progenitor cells. Glast-Cre ERT2 ;tdT (Ctrl), Glast-Cre ERT2 ;Rbpj flox/wt ;tdT and Glast-Cre ERT2 ;Rbpj flox/flox ;tdT mice were received TAM injection at postnatal day 1 (P1) and harvested at P12. (c) Representative immunostaining of Otx2 and Sox9 on retinal. (d) Percentage of tdT+ rod photoreceptor cells in overall tdT+ cells. (e) Percentage of tdT+ Sox9+ cells in overall tdT+ cells. n≥3 mice, ns. not significant, **** P < 0.0001, by one-way ANOVA with Tukey post hoc test.

    Article Snippet: Rbpj flox/flox mice (strain: Rbpj em2Lutzy ), tdTomato reporter mice (strain B6.Cg-Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze /J ) , Sun1:GFP reporter mice (Strain B6.129-Gt(ROSA)26Sor tm5.1(CAG-Sun1/sfGFP)Nat /MmbeJ ) , and Glast-Cre ERT2 reporter mice (strain Tg(Slc1a3-cre/ERT)1Nat/J )( ) mice were purchased from the Jackson Laboratory.

    Techniques: Injection, Immunostaining

    (a) Representative immunostaining of mCAR and Sox2 on retinal sections from Glast-Cre ERT2 ;tdT (Ctrl), Glast-Cre ERT2 ;Rbpj flox/wt ;tdT and Glast-Cre ERT2 ;Rbpj flox/flox ;tdT mice received tamoxifen (TAM) injection at postnatal day 1 (P1) and harvested at P12. (b) Magnified views of the highlighted regions in (a). (c) Number of cone photoreceptor cells in 250μm. n≥3 mice, data are presented as mean ± SEM. ns=not significant, by one-way ANOVA with Tukey’s post hoc test.

    Journal: bioRxiv

    Article Title: Synergistic Inhibition of Notch Signaling and Forced Cell Cycle Re-entry Drive Müller Glia Reprogramming in Uninjured Mouse Retina

    doi: 10.64898/2026.03.09.710684

    Figure Lengend Snippet: (a) Representative immunostaining of mCAR and Sox2 on retinal sections from Glast-Cre ERT2 ;tdT (Ctrl), Glast-Cre ERT2 ;Rbpj flox/wt ;tdT and Glast-Cre ERT2 ;Rbpj flox/flox ;tdT mice received tamoxifen (TAM) injection at postnatal day 1 (P1) and harvested at P12. (b) Magnified views of the highlighted regions in (a). (c) Number of cone photoreceptor cells in 250μm. n≥3 mice, data are presented as mean ± SEM. ns=not significant, by one-way ANOVA with Tukey’s post hoc test.

    Article Snippet: Rbpj flox/flox mice (strain: Rbpj em2Lutzy ), tdTomato reporter mice (strain B6.Cg-Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze /J ) , Sun1:GFP reporter mice (Strain B6.129-Gt(ROSA)26Sor tm5.1(CAG-Sun1/sfGFP)Nat /MmbeJ ) , and Glast-Cre ERT2 reporter mice (strain Tg(Slc1a3-cre/ERT)1Nat/J )( ) mice were purchased from the Jackson Laboratory.

    Techniques: Immunostaining, Injection

    (a) Representative immunostaining of Otx2 on retinal sections from Glast-Cre ERT2 ;tdT (Ctrl), Glast-Cre ERT2 ;Rbpj flox/wt ;tdT and Glast-Cre ERT2 ;Rbpj flox/flox ;tdT mice received tamoxifen (TAM) injection at postnatal day 1 (P1) and harvested at P12. (b) Magnified views of the highlighted regions in (a). (c) Percentage of tdT+ Otx2+ cells in overall tdT+ cells. n≥3 mice, ns=not significant, * P < 0.05, ** P < 0.01, by one-way ANOVA with Tukey post hoc test.

    Journal: bioRxiv

    Article Title: Synergistic Inhibition of Notch Signaling and Forced Cell Cycle Re-entry Drive Müller Glia Reprogramming in Uninjured Mouse Retina

    doi: 10.64898/2026.03.09.710684

    Figure Lengend Snippet: (a) Representative immunostaining of Otx2 on retinal sections from Glast-Cre ERT2 ;tdT (Ctrl), Glast-Cre ERT2 ;Rbpj flox/wt ;tdT and Glast-Cre ERT2 ;Rbpj flox/flox ;tdT mice received tamoxifen (TAM) injection at postnatal day 1 (P1) and harvested at P12. (b) Magnified views of the highlighted regions in (a). (c) Percentage of tdT+ Otx2+ cells in overall tdT+ cells. n≥3 mice, ns=not significant, * P < 0.05, ** P < 0.01, by one-way ANOVA with Tukey post hoc test.

    Article Snippet: Rbpj flox/flox mice (strain: Rbpj em2Lutzy ), tdTomato reporter mice (strain B6.Cg-Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze /J ) , Sun1:GFP reporter mice (Strain B6.129-Gt(ROSA)26Sor tm5.1(CAG-Sun1/sfGFP)Nat /MmbeJ ) , and Glast-Cre ERT2 reporter mice (strain Tg(Slc1a3-cre/ERT)1Nat/J )( ) mice were purchased from the Jackson Laboratory.

    Techniques: Immunostaining, Injection

    (a) Representative immunostaining of HuC/D and Pax6 on retinal sections from Glast-Cre ERT2 ;tdT (Ctrl), Glast-Cre ERT2 ;Rbpj flox/wt ;tdT and Glast-Cre ERT2 ;Rbpj flox/flox ;tdT mice received TAM injection at postnatal day 1 (P1) and harvested at P12. (b) Magnified views of the highlighted regions in (a). (c) Percentage of tdT+ HuC/D+ Pax6+ cells in overall tdT+ cells. n≥3 mice, data are presented as mean ± SEM. ns=not significant, by one-way ANOVA with Tukey’s post hoc test.

    Journal: bioRxiv

    Article Title: Synergistic Inhibition of Notch Signaling and Forced Cell Cycle Re-entry Drive Müller Glia Reprogramming in Uninjured Mouse Retina

    doi: 10.64898/2026.03.09.710684

    Figure Lengend Snippet: (a) Representative immunostaining of HuC/D and Pax6 on retinal sections from Glast-Cre ERT2 ;tdT (Ctrl), Glast-Cre ERT2 ;Rbpj flox/wt ;tdT and Glast-Cre ERT2 ;Rbpj flox/flox ;tdT mice received TAM injection at postnatal day 1 (P1) and harvested at P12. (b) Magnified views of the highlighted regions in (a). (c) Percentage of tdT+ HuC/D+ Pax6+ cells in overall tdT+ cells. n≥3 mice, data are presented as mean ± SEM. ns=not significant, by one-way ANOVA with Tukey’s post hoc test.

    Article Snippet: Rbpj flox/flox mice (strain: Rbpj em2Lutzy ), tdTomato reporter mice (strain B6.Cg-Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze /J ) , Sun1:GFP reporter mice (Strain B6.129-Gt(ROSA)26Sor tm5.1(CAG-Sun1/sfGFP)Nat /MmbeJ ) , and Glast-Cre ERT2 reporter mice (strain Tg(Slc1a3-cre/ERT)1Nat/J )( ) mice were purchased from the Jackson Laboratory.

    Techniques: Immunostaining, Injection

    (a) Representative immunostaining of Rbpms on retinal sections from Glast-Cre ERT2 ;tdT (Ctrl), Glast-Cre ERT2 ;Rbpj flox/wt ;tdT and Glast-Cre ERT2 ;Rbpj flox/flox ;tdT mice received tamoxifen (TAM) injection at P1 and harvested at P12. (b) Magnified views of the highlighted regions in (a). (c) Number of Rbpms+ cells per 150μm. n≥3 mice, data are presented as mean ± SEM. ns=not significant, by one-way ANOVA with Tukey’s post hoc test.

    Journal: bioRxiv

    Article Title: Synergistic Inhibition of Notch Signaling and Forced Cell Cycle Re-entry Drive Müller Glia Reprogramming in Uninjured Mouse Retina

    doi: 10.64898/2026.03.09.710684

    Figure Lengend Snippet: (a) Representative immunostaining of Rbpms on retinal sections from Glast-Cre ERT2 ;tdT (Ctrl), Glast-Cre ERT2 ;Rbpj flox/wt ;tdT and Glast-Cre ERT2 ;Rbpj flox/flox ;tdT mice received tamoxifen (TAM) injection at P1 and harvested at P12. (b) Magnified views of the highlighted regions in (a). (c) Number of Rbpms+ cells per 150μm. n≥3 mice, data are presented as mean ± SEM. ns=not significant, by one-way ANOVA with Tukey’s post hoc test.

    Article Snippet: Rbpj flox/flox mice (strain: Rbpj em2Lutzy ), tdTomato reporter mice (strain B6.Cg-Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze /J ) , Sun1:GFP reporter mice (Strain B6.129-Gt(ROSA)26Sor tm5.1(CAG-Sun1/sfGFP)Nat /MmbeJ ) , and Glast-Cre ERT2 reporter mice (strain Tg(Slc1a3-cre/ERT)1Nat/J )( ) mice were purchased from the Jackson Laboratory.

    Techniques: Immunostaining, Injection

    (a) Schematic illustration of MG dedifferentiation and reprogramming experiment. (b) Representative immunostaining of Sox9 on retinal sections from Glast-Cre ERT2 ;Sun1:GFP and Glast-Cre ERT2 ;Rbpj flox/flox ;Sun1:GFP mice in different timepoints post TAM injection. The white arrows refer to GFP+ Sox9- cells. (c) Magnified views of the highlighted regions in (b). (d) Percentage of GFP+ Sox9- cells in overall GFP+ cells. n=3 mice, data are presented as mean ± SEM. ns=not significant, **** P < 0.0001, by one-way ANOVA with Tukey’s post hoc test. (e) Percentage of GFP+ Otx2+ cells in overall GFP+ cells. n=3 mice, data are presented as mean ± SEM. ns=not significant, ** P < 0.01, by one-way ANOVA with Tukey’s post hoc test.

    Journal: bioRxiv

    Article Title: Synergistic Inhibition of Notch Signaling and Forced Cell Cycle Re-entry Drive Müller Glia Reprogramming in Uninjured Mouse Retina

    doi: 10.64898/2026.03.09.710684

    Figure Lengend Snippet: (a) Schematic illustration of MG dedifferentiation and reprogramming experiment. (b) Representative immunostaining of Sox9 on retinal sections from Glast-Cre ERT2 ;Sun1:GFP and Glast-Cre ERT2 ;Rbpj flox/flox ;Sun1:GFP mice in different timepoints post TAM injection. The white arrows refer to GFP+ Sox9- cells. (c) Magnified views of the highlighted regions in (b). (d) Percentage of GFP+ Sox9- cells in overall GFP+ cells. n=3 mice, data are presented as mean ± SEM. ns=not significant, **** P < 0.0001, by one-way ANOVA with Tukey’s post hoc test. (e) Percentage of GFP+ Otx2+ cells in overall GFP+ cells. n=3 mice, data are presented as mean ± SEM. ns=not significant, ** P < 0.01, by one-way ANOVA with Tukey’s post hoc test.

    Article Snippet: Rbpj flox/flox mice (strain: Rbpj em2Lutzy ), tdTomato reporter mice (strain B6.Cg-Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze /J ) , Sun1:GFP reporter mice (Strain B6.129-Gt(ROSA)26Sor tm5.1(CAG-Sun1/sfGFP)Nat /MmbeJ ) , and Glast-Cre ERT2 reporter mice (strain Tg(Slc1a3-cre/ERT)1Nat/J )( ) mice were purchased from the Jackson Laboratory.

    Techniques: Immunostaining, Injection

    (a) Schematic illustration of MG dedifferentiation and reprogramming experiment. (b) Representative immunostaining of Sox9 on retinal sections from Glast-Cre ERT2 ;Sun1:GFP and Glast-Cre ERT2 ;Rbpj flox/flox ;Sun1:GFP mice harvested at different timepoints post TAM injection. The white arrows refer to GFP+ Sox9- cells. (c) Magnified views of the highlighted regions in (B).

    Journal: bioRxiv

    Article Title: Synergistic Inhibition of Notch Signaling and Forced Cell Cycle Re-entry Drive Müller Glia Reprogramming in Uninjured Mouse Retina

    doi: 10.64898/2026.03.09.710684

    Figure Lengend Snippet: (a) Schematic illustration of MG dedifferentiation and reprogramming experiment. (b) Representative immunostaining of Sox9 on retinal sections from Glast-Cre ERT2 ;Sun1:GFP and Glast-Cre ERT2 ;Rbpj flox/flox ;Sun1:GFP mice harvested at different timepoints post TAM injection. The white arrows refer to GFP+ Sox9- cells. (c) Magnified views of the highlighted regions in (B).

    Article Snippet: Rbpj flox/flox mice (strain: Rbpj em2Lutzy ), tdTomato reporter mice (strain B6.Cg-Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze /J ) , Sun1:GFP reporter mice (Strain B6.129-Gt(ROSA)26Sor tm5.1(CAG-Sun1/sfGFP)Nat /MmbeJ ) , and Glast-Cre ERT2 reporter mice (strain Tg(Slc1a3-cre/ERT)1Nat/J )( ) mice were purchased from the Jackson Laboratory.

    Techniques: Immunostaining, Injection

    (a) Schematic illustration of the experiment examining MG proliferation. (b) Representative immunostaining of Otx2 and EdU on retinal sections from Glast-Cre ERT2 ;Sun1:GFP and Glast-Cre ERT2 ;Rbpj flox/flox ;Sun1:GFP mice harvested at 4 months post TAM injection.

    Journal: bioRxiv

    Article Title: Synergistic Inhibition of Notch Signaling and Forced Cell Cycle Re-entry Drive Müller Glia Reprogramming in Uninjured Mouse Retina

    doi: 10.64898/2026.03.09.710684

    Figure Lengend Snippet: (a) Schematic illustration of the experiment examining MG proliferation. (b) Representative immunostaining of Otx2 and EdU on retinal sections from Glast-Cre ERT2 ;Sun1:GFP and Glast-Cre ERT2 ;Rbpj flox/flox ;Sun1:GFP mice harvested at 4 months post TAM injection.

    Article Snippet: Rbpj flox/flox mice (strain: Rbpj em2Lutzy ), tdTomato reporter mice (strain B6.Cg-Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze /J ) , Sun1:GFP reporter mice (Strain B6.129-Gt(ROSA)26Sor tm5.1(CAG-Sun1/sfGFP)Nat /MmbeJ ) , and Glast-Cre ERT2 reporter mice (strain Tg(Slc1a3-cre/ERT)1Nat/J )( ) mice were purchased from the Jackson Laboratory.

    Techniques: Immunostaining, Injection

    (a) Schematic illustration of proliferation level comparison experiment. (b) Representative immunostaining of EdU on retinal sections from Glast-Cre ERT2 ;tdT (Ctrl), Glast-Cre ERT2 ;Rbpj flox/wt ;tdT and Glast-Cre ERT2 ;Rbpj flox/flox ;tdT mice received CCA injection. (c) Number of EdU+ cells in 500μm. n≥3 mice, data are presented as mean ± SEM. ns=not significant, *** P < 0.001; **** P < 0.0001, by one-way ANOVA with Tukey’s post hoc test. (d-e) Schematic illustration of the comparison of proliferation levels between different CCA injection and TAM administration orders. (f) Representative immunostaining of EdU on retinal sections from Glast-Cre ERT2 ;Rbpj flox/flox ;tdT mice received CCA injection. (g) Number of EdU+ cells in 500μm. n=3 mice, data are presented as mean ± SEM. ns=not significant, **** P < 0.0001, by one-way ANOVA with Tukey’s post hoc test.

    Journal: bioRxiv

    Article Title: Synergistic Inhibition of Notch Signaling and Forced Cell Cycle Re-entry Drive Müller Glia Reprogramming in Uninjured Mouse Retina

    doi: 10.64898/2026.03.09.710684

    Figure Lengend Snippet: (a) Schematic illustration of proliferation level comparison experiment. (b) Representative immunostaining of EdU on retinal sections from Glast-Cre ERT2 ;tdT (Ctrl), Glast-Cre ERT2 ;Rbpj flox/wt ;tdT and Glast-Cre ERT2 ;Rbpj flox/flox ;tdT mice received CCA injection. (c) Number of EdU+ cells in 500μm. n≥3 mice, data are presented as mean ± SEM. ns=not significant, *** P < 0.001; **** P < 0.0001, by one-way ANOVA with Tukey’s post hoc test. (d-e) Schematic illustration of the comparison of proliferation levels between different CCA injection and TAM administration orders. (f) Representative immunostaining of EdU on retinal sections from Glast-Cre ERT2 ;Rbpj flox/flox ;tdT mice received CCA injection. (g) Number of EdU+ cells in 500μm. n=3 mice, data are presented as mean ± SEM. ns=not significant, **** P < 0.0001, by one-way ANOVA with Tukey’s post hoc test.

    Article Snippet: Rbpj flox/flox mice (strain: Rbpj em2Lutzy ), tdTomato reporter mice (strain B6.Cg-Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze /J ) , Sun1:GFP reporter mice (Strain B6.129-Gt(ROSA)26Sor tm5.1(CAG-Sun1/sfGFP)Nat /MmbeJ ) , and Glast-Cre ERT2 reporter mice (strain Tg(Slc1a3-cre/ERT)1Nat/J )( ) mice were purchased from the Jackson Laboratory.

    Techniques: Comparison, Immunostaining, Injection

    (a) Schematic illustration of MG dedifferentiation experiment. (b) Representative immunostaining of Sox9 on retinal sections from Glast-Cre ERT2 ;Sun1:GFP (Ctrl) and Glast-Cre ERT2 ;Rbpj flox/flox ;Sun1:GFP mice at 3 weeks and 4 months post CCA treatment. The white arrows refer to GFP+ Sox9- cells. (c) Magnified views of the highlighted regions in (b). (d) Percentage of GFP+ Sox9- cells in overall GFP+ cells. 3W: 3 weeks, 4M: 4 months, n=3 mice, data are presented as mean ± SEM. ns=not significant, **** P < 0.0001, by one-way ANOVA with Tukey’s post hoc test. (e) Percentage of GFP⁺ Sox9⁻ cells among total GFP⁺ cells in the 4-month samples. n=3 mice, data are presented as mean ± SEM. ns=not significant, * P < 0.05, **** P < 0.0001, by one-way ANOVA with Tukey’s post hoc test. (f) Number of Sox9+ cells per 500μm in the 4-month samples. n=3 mice, data are presented as mean ± SEM. ns=not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, by one-way ANOVA with Tukey’s post hoc test.

    Journal: bioRxiv

    Article Title: Synergistic Inhibition of Notch Signaling and Forced Cell Cycle Re-entry Drive Müller Glia Reprogramming in Uninjured Mouse Retina

    doi: 10.64898/2026.03.09.710684

    Figure Lengend Snippet: (a) Schematic illustration of MG dedifferentiation experiment. (b) Representative immunostaining of Sox9 on retinal sections from Glast-Cre ERT2 ;Sun1:GFP (Ctrl) and Glast-Cre ERT2 ;Rbpj flox/flox ;Sun1:GFP mice at 3 weeks and 4 months post CCA treatment. The white arrows refer to GFP+ Sox9- cells. (c) Magnified views of the highlighted regions in (b). (d) Percentage of GFP+ Sox9- cells in overall GFP+ cells. 3W: 3 weeks, 4M: 4 months, n=3 mice, data are presented as mean ± SEM. ns=not significant, **** P < 0.0001, by one-way ANOVA with Tukey’s post hoc test. (e) Percentage of GFP⁺ Sox9⁻ cells among total GFP⁺ cells in the 4-month samples. n=3 mice, data are presented as mean ± SEM. ns=not significant, * P < 0.05, **** P < 0.0001, by one-way ANOVA with Tukey’s post hoc test. (f) Number of Sox9+ cells per 500μm in the 4-month samples. n=3 mice, data are presented as mean ± SEM. ns=not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, by one-way ANOVA with Tukey’s post hoc test.

    Article Snippet: Rbpj flox/flox mice (strain: Rbpj em2Lutzy ), tdTomato reporter mice (strain B6.Cg-Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze /J ) , Sun1:GFP reporter mice (Strain B6.129-Gt(ROSA)26Sor tm5.1(CAG-Sun1/sfGFP)Nat /MmbeJ ) , and Glast-Cre ERT2 reporter mice (strain Tg(Slc1a3-cre/ERT)1Nat/J )( ) mice were purchased from the Jackson Laboratory.

    Techniques: Immunostaining

    (a) Schematic illustration of the snRNA-seq experiment. To induce Rbpj deletion and GFP expression in MG, we administered TAM for 7 consecutive days in Glast-Cre ERT2 ;Sun1:GFP and Glast-Cre ERT2 ;Rbpj flox/flox ;Sun1:GFP mice from P28. At P36, CCA was injected into the CCA and Rbpj KO+CCA groups, while control and Rbpj KO mice were uninjected by any AAV vector. 3-4 retinas from mice at indicated ages were pooled together for nuclei extraction, and MG nuclei were then isolated by GFP signal via FACS for snRNA-seq. (b) UMAP plot of snRNA-seq data from four treatment groups (Ctrl, CCA, Rbpj KO, Rbpj KO+CCA) at different timepoints, and separated UMAP based on different timepoints. clusters were identified based on known marker gene expression. (c) Proportions of cell clusters within Ctrl, CCA, Rbpj KO and + groups at different timepoints. (d) Feature plots highlighting the cluster of quiescent MG ( Kcnj10 ), proliferating MG ( Mki67 ), reactivated MG ( Gfap ), MGPC ( Dll1 , Ascl1 ), transitional MG ( Mt-Nd4 ), AC-like MG ( Elavl3 ), and BC-like MG ( Otx2 ). (e) Heatmap showing the expression of top 50 differentially expressed genes (DEGs) of 3-week reactivated MG between CCA and Rbpj KO+CCA (p<0.05). (f) Heatmap showing the expression of top 50 DEGs of 4-month MGPC between Rbpj KO and Rbpj KO+CCA (p<0.05).

    Journal: bioRxiv

    Article Title: Synergistic Inhibition of Notch Signaling and Forced Cell Cycle Re-entry Drive Müller Glia Reprogramming in Uninjured Mouse Retina

    doi: 10.64898/2026.03.09.710684

    Figure Lengend Snippet: (a) Schematic illustration of the snRNA-seq experiment. To induce Rbpj deletion and GFP expression in MG, we administered TAM for 7 consecutive days in Glast-Cre ERT2 ;Sun1:GFP and Glast-Cre ERT2 ;Rbpj flox/flox ;Sun1:GFP mice from P28. At P36, CCA was injected into the CCA and Rbpj KO+CCA groups, while control and Rbpj KO mice were uninjected by any AAV vector. 3-4 retinas from mice at indicated ages were pooled together for nuclei extraction, and MG nuclei were then isolated by GFP signal via FACS for snRNA-seq. (b) UMAP plot of snRNA-seq data from four treatment groups (Ctrl, CCA, Rbpj KO, Rbpj KO+CCA) at different timepoints, and separated UMAP based on different timepoints. clusters were identified based on known marker gene expression. (c) Proportions of cell clusters within Ctrl, CCA, Rbpj KO and + groups at different timepoints. (d) Feature plots highlighting the cluster of quiescent MG ( Kcnj10 ), proliferating MG ( Mki67 ), reactivated MG ( Gfap ), MGPC ( Dll1 , Ascl1 ), transitional MG ( Mt-Nd4 ), AC-like MG ( Elavl3 ), and BC-like MG ( Otx2 ). (e) Heatmap showing the expression of top 50 differentially expressed genes (DEGs) of 3-week reactivated MG between CCA and Rbpj KO+CCA (p<0.05). (f) Heatmap showing the expression of top 50 DEGs of 4-month MGPC between Rbpj KO and Rbpj KO+CCA (p<0.05).

    Article Snippet: Rbpj flox/flox mice (strain: Rbpj em2Lutzy ), tdTomato reporter mice (strain B6.Cg-Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze /J ) , Sun1:GFP reporter mice (Strain B6.129-Gt(ROSA)26Sor tm5.1(CAG-Sun1/sfGFP)Nat /MmbeJ ) , and Glast-Cre ERT2 reporter mice (strain Tg(Slc1a3-cre/ERT)1Nat/J )( ) mice were purchased from the Jackson Laboratory.

    Techniques: Expressing, Injection, Control, Plasmid Preparation, Extraction, Isolation, Marker, Gene Expression

    (a) Dot plot showing gene expression and cell percentages for quiescent MG, proliferating MG, reactivated MG, MGPC, transitional MG, AC-like MG, BC-like MG. (b) Separated UMAP plot of snRNA-seq data of Ctrl, CCA, Rbpj KO, Rbpj KO+CCA treatment. (c) Separated proportions of cell clusters within Ctrl, CCA, Rbpj KO and Rbpj KO+CCA groups at different timepoints.

    Journal: bioRxiv

    Article Title: Synergistic Inhibition of Notch Signaling and Forced Cell Cycle Re-entry Drive Müller Glia Reprogramming in Uninjured Mouse Retina

    doi: 10.64898/2026.03.09.710684

    Figure Lengend Snippet: (a) Dot plot showing gene expression and cell percentages for quiescent MG, proliferating MG, reactivated MG, MGPC, transitional MG, AC-like MG, BC-like MG. (b) Separated UMAP plot of snRNA-seq data of Ctrl, CCA, Rbpj KO, Rbpj KO+CCA treatment. (c) Separated proportions of cell clusters within Ctrl, CCA, Rbpj KO and Rbpj KO+CCA groups at different timepoints.

    Article Snippet: Rbpj flox/flox mice (strain: Rbpj em2Lutzy ), tdTomato reporter mice (strain B6.Cg-Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze /J ) , Sun1:GFP reporter mice (Strain B6.129-Gt(ROSA)26Sor tm5.1(CAG-Sun1/sfGFP)Nat /MmbeJ ) , and Glast-Cre ERT2 reporter mice (strain Tg(Slc1a3-cre/ERT)1Nat/J )( ) mice were purchased from the Jackson Laboratory.

    Techniques: Gene Expression

    (a) The split UMAP by the condition of time and treatment. To monitor the progress of MG regeneration, we implemented three time points (1 week (1W), 3 weeks (3W), 4 months (4M)) and four treatment groups (Ctrl, CCA, Rbpj KO, Rbpj KO+CCA).

    Journal: bioRxiv

    Article Title: Synergistic Inhibition of Notch Signaling and Forced Cell Cycle Re-entry Drive Müller Glia Reprogramming in Uninjured Mouse Retina

    doi: 10.64898/2026.03.09.710684

    Figure Lengend Snippet: (a) The split UMAP by the condition of time and treatment. To monitor the progress of MG regeneration, we implemented three time points (1 week (1W), 3 weeks (3W), 4 months (4M)) and four treatment groups (Ctrl, CCA, Rbpj KO, Rbpj KO+CCA).

    Article Snippet: Rbpj flox/flox mice (strain: Rbpj em2Lutzy ), tdTomato reporter mice (strain B6.Cg-Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze /J ) , Sun1:GFP reporter mice (Strain B6.129-Gt(ROSA)26Sor tm5.1(CAG-Sun1/sfGFP)Nat /MmbeJ ) , and Glast-Cre ERT2 reporter mice (strain Tg(Slc1a3-cre/ERT)1Nat/J )( ) mice were purchased from the Jackson Laboratory.

    Techniques:

    (a) Subclusters of the BC-like population. The BC-like MG (outlined in red) was used for subclustering analysis. (b) Feature plot of BC-like subtypes showing the cluster of OFF-cone BC ( Pcdh17 , Zfhx4 ), ON-cone BC ( Isl1 , Grm6 ) and rod BC ( Isl1 , Prkca , Cep112 ). (c) Dot plot showing gene expression and cell percentages for OFF-cone BC, ON-cone BC and rod BC. (d) Percentage of BC subtypes in different treatment groups. (e-f) Pcdh17 and Grm6 mRNA in situ hybridization in the Ctrl and Rbpj KO+OCCA groups. The white dashed boxes indicate the position of the enlarged images. (g-h) Representative immunostaining of EdU and PKCα on retinal sections of the Ctrl and Rbpj KO+CCA samples. The white dashed boxes indicate the position of the enlarged images. (i) Percentage showing GFP+ Grm6 + cells in overall GFP+ cells, GFP+ Pcdh17 + cells in overall GFP+ cells and GFP+ PKCα+ cells in overall GFP+ cells in the Ctrl and Rbpj KO+CCA groups. n=3 mice, * P < 0.05, ** P < 0.01, **** P < 0.0001, by unpaired two-tailed student’s t-test. (j) Representative immunostaining of MG-derived cells with bipolar cell morphology.

    Journal: bioRxiv

    Article Title: Synergistic Inhibition of Notch Signaling and Forced Cell Cycle Re-entry Drive Müller Glia Reprogramming in Uninjured Mouse Retina

    doi: 10.64898/2026.03.09.710684

    Figure Lengend Snippet: (a) Subclusters of the BC-like population. The BC-like MG (outlined in red) was used for subclustering analysis. (b) Feature plot of BC-like subtypes showing the cluster of OFF-cone BC ( Pcdh17 , Zfhx4 ), ON-cone BC ( Isl1 , Grm6 ) and rod BC ( Isl1 , Prkca , Cep112 ). (c) Dot plot showing gene expression and cell percentages for OFF-cone BC, ON-cone BC and rod BC. (d) Percentage of BC subtypes in different treatment groups. (e-f) Pcdh17 and Grm6 mRNA in situ hybridization in the Ctrl and Rbpj KO+OCCA groups. The white dashed boxes indicate the position of the enlarged images. (g-h) Representative immunostaining of EdU and PKCα on retinal sections of the Ctrl and Rbpj KO+CCA samples. The white dashed boxes indicate the position of the enlarged images. (i) Percentage showing GFP+ Grm6 + cells in overall GFP+ cells, GFP+ Pcdh17 + cells in overall GFP+ cells and GFP+ PKCα+ cells in overall GFP+ cells in the Ctrl and Rbpj KO+CCA groups. n=3 mice, * P < 0.05, ** P < 0.01, **** P < 0.0001, by unpaired two-tailed student’s t-test. (j) Representative immunostaining of MG-derived cells with bipolar cell morphology.

    Article Snippet: Rbpj flox/flox mice (strain: Rbpj em2Lutzy ), tdTomato reporter mice (strain B6.Cg-Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze /J ) , Sun1:GFP reporter mice (Strain B6.129-Gt(ROSA)26Sor tm5.1(CAG-Sun1/sfGFP)Nat /MmbeJ ) , and Glast-Cre ERT2 reporter mice (strain Tg(Slc1a3-cre/ERT)1Nat/J )( ) mice were purchased from the Jackson Laboratory.

    Techniques: Gene Expression, In Situ Hybridization, Immunostaining, Two Tailed Test, Derivative Assay

    (a) Schematic illustration of the snATAC-seq experiment. (b) Proportions of cell clusters within Ctrl, CCA, Rbpj KO and Rbpj KO+CCA groups. (c) UMAP plot of snATAC-seq data from four treatment groups (Ctrl, CCA, Rbpj KO, Rbpj KO+CCA). (d) Feature plots highlighting the cluster of quiescent MG (Kcnj10), KO MG (Hes5), active MG (Bal3), MGPC (Dll1), AC-like MG (Caln1), BC-like MG (Otx2). (e) Dot plot showing gene expression and cell percentages for quiescent MG, KO MG, active MG, MGPC, AC-like MG, BC-like MG. (f) Volcano plot showing the differential gene expression of MG and active MG within CCA treatment group (p<0.05). Red dots indicate the genes upregulated in active MG and blue dots indicate genes downregulated in active MG. (g) WikiPathways enrichment analysis of upregulated genes in active MG (p<0.05). (h) Increased chromatin accessibility at the Neurod2 locus was observed in the active MG. The black arrow shows the transcription direction.

    Journal: bioRxiv

    Article Title: Synergistic Inhibition of Notch Signaling and Forced Cell Cycle Re-entry Drive Müller Glia Reprogramming in Uninjured Mouse Retina

    doi: 10.64898/2026.03.09.710684

    Figure Lengend Snippet: (a) Schematic illustration of the snATAC-seq experiment. (b) Proportions of cell clusters within Ctrl, CCA, Rbpj KO and Rbpj KO+CCA groups. (c) UMAP plot of snATAC-seq data from four treatment groups (Ctrl, CCA, Rbpj KO, Rbpj KO+CCA). (d) Feature plots highlighting the cluster of quiescent MG (Kcnj10), KO MG (Hes5), active MG (Bal3), MGPC (Dll1), AC-like MG (Caln1), BC-like MG (Otx2). (e) Dot plot showing gene expression and cell percentages for quiescent MG, KO MG, active MG, MGPC, AC-like MG, BC-like MG. (f) Volcano plot showing the differential gene expression of MG and active MG within CCA treatment group (p<0.05). Red dots indicate the genes upregulated in active MG and blue dots indicate genes downregulated in active MG. (g) WikiPathways enrichment analysis of upregulated genes in active MG (p<0.05). (h) Increased chromatin accessibility at the Neurod2 locus was observed in the active MG. The black arrow shows the transcription direction.

    Article Snippet: Rbpj flox/flox mice (strain: Rbpj em2Lutzy ), tdTomato reporter mice (strain B6.Cg-Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze /J ) , Sun1:GFP reporter mice (Strain B6.129-Gt(ROSA)26Sor tm5.1(CAG-Sun1/sfGFP)Nat /MmbeJ ) , and Glast-Cre ERT2 reporter mice (strain Tg(Slc1a3-cre/ERT)1Nat/J )( ) mice were purchased from the Jackson Laboratory.

    Techniques: Gene Expression

    (a) Schematic illustration of ZO1 staining experiment. (b) Representative immunostaining of Otx2 and ZO1 on retinal sections from Glast-Cre ERT2 ;tdT and Glast-Cre ERT2 ;Rbpj flox/flox ;tdT mice at 9 months post TAM injection. The white arrows refer to tdT+ Otx2+ cells.

    Journal: bioRxiv

    Article Title: Synergistic Inhibition of Notch Signaling and Forced Cell Cycle Re-entry Drive Müller Glia Reprogramming in Uninjured Mouse Retina

    doi: 10.64898/2026.03.09.710684

    Figure Lengend Snippet: (a) Schematic illustration of ZO1 staining experiment. (b) Representative immunostaining of Otx2 and ZO1 on retinal sections from Glast-Cre ERT2 ;tdT and Glast-Cre ERT2 ;Rbpj flox/flox ;tdT mice at 9 months post TAM injection. The white arrows refer to tdT+ Otx2+ cells.

    Article Snippet: Rbpj flox/flox mice (strain: Rbpj em2Lutzy ), tdTomato reporter mice (strain B6.Cg-Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze /J ) , Sun1:GFP reporter mice (Strain B6.129-Gt(ROSA)26Sor tm5.1(CAG-Sun1/sfGFP)Nat /MmbeJ ) , and Glast-Cre ERT2 reporter mice (strain Tg(Slc1a3-cre/ERT)1Nat/J )( ) mice were purchased from the Jackson Laboratory.

    Techniques: Staining, Immunostaining, Injection

    miR-UL36 impairs Notch signaling through targeting Notch3 and RBPJ. ( A ) HEK293 cells were transfected with a 4× CSL-Luciferase construct as well as a plasmid expressing the intracellular domain of Notch3 and either negative control miRNA mimic, miR-US33, miR-UL36, or miR-UL112. Protein lysates were harvested 24 h post-transfection, and luciferase expression was monitored using a luminometer ( n = 3; P < 0.05 calculated using a one-sample t and Wilcoxon test). ( B and C ) NHDFs were transfected with negative control miRNA mimic, miR-UL36, or siRNA against Notch3 ( B ) or RBPJ ( C ). Protein lysates were harvested 48 h post-transfection, and immunoblots were performed using the indicated antibodies. Densitometry was performed using ImageJ software for three independent experiments and plotted in the right-hand panels. ( D and E ). NHDF were mock-infected or infected with WT HCMV or a miR-UL36 mutant virus for 48 h. Protein lysates were harvested, and immunoblotting was performed using the indicated antibodies. Densitometry was performed using ImageJ software for three independent experiments and plotted in the right-hand panels. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 as determined by two-tailed t -test. ( F ) RNA was harvested from cells infected as in ( D and E ) and subjected to qRT-PCR using primers for the Notch target genes HES1 and HEY1. ( n = 3; * P < 0.05 by two-tailed t -test).

    Journal: mBio

    Article Title: HCMV promotes viral reactivation through the coordinated regulation of Notch signaling by UL8 and miR-UL36

    doi: 10.1128/mbio.03377-25

    Figure Lengend Snippet: miR-UL36 impairs Notch signaling through targeting Notch3 and RBPJ. ( A ) HEK293 cells were transfected with a 4× CSL-Luciferase construct as well as a plasmid expressing the intracellular domain of Notch3 and either negative control miRNA mimic, miR-US33, miR-UL36, or miR-UL112. Protein lysates were harvested 24 h post-transfection, and luciferase expression was monitored using a luminometer ( n = 3; P < 0.05 calculated using a one-sample t and Wilcoxon test). ( B and C ) NHDFs were transfected with negative control miRNA mimic, miR-UL36, or siRNA against Notch3 ( B ) or RBPJ ( C ). Protein lysates were harvested 48 h post-transfection, and immunoblots were performed using the indicated antibodies. Densitometry was performed using ImageJ software for three independent experiments and plotted in the right-hand panels. ( D and E ). NHDF were mock-infected or infected with WT HCMV or a miR-UL36 mutant virus for 48 h. Protein lysates were harvested, and immunoblotting was performed using the indicated antibodies. Densitometry was performed using ImageJ software for three independent experiments and plotted in the right-hand panels. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 as determined by two-tailed t -test. ( F ) RNA was harvested from cells infected as in ( D and E ) and subjected to qRT-PCR using primers for the Notch target genes HES1 and HEY1. ( n = 3; * P < 0.05 by two-tailed t -test).

    Article Snippet: The following commercial antibodies were used: Notch3 (2889, Cell Signaling), RBPJ (5442, Cell Signaling), GAPDH (ab8245, Abcam), HCMV IE2 (MAB810, Sigma Aldrich), UL44 (Virusys Corporation, P1202-2), and Turbo ID (AS204440, Agrisera).

    Techniques: Transfection, Luciferase, Construct, Plasmid Preparation, Expressing, Negative Control, Western Blot, Software, Infection, Mutagenesis, Virus, Two Tailed Test, Quantitative RT-PCR

    Nitrate functions by Notch pathway inhibition positively transcripting TFF2 in vitro. (A) Diagram of the wild‐type and mutant sequences for the three predicted RBPJ binding sites within the 251 bp TFF2 promoter probes. (B) Representative electrophoretic mobility shift assay (EMSA) autoradiograph. Hot probe is the biotin‐labeled wild‐type oligonucleotides of the truncated TFF2 promoter containing the binding motif; Mutant probe is the labeled oligonucleotides sequence with nucleotides mutated. The cold probe is nonlabeled competitive wild‐type probes (100 and 50 that of the concentrations). The shifted bands are indicated by arrows, which suggested the formation of DNA–protein complexes (lane 2, 3, 7). The super shifted bands indicated the formation of DNA–protein–antibody complexes (lane 3, 7). “+” and “−” represent presence and absence, respectively. (C) Relative TFF2 promoter (Full, Mut1, Mut2, and Mut3) luciferase activity was detected by DLR assays in RBPJ overexpressed and normal‐expressed GES‐1 cells. (D) A schematic diagram showing the location of RBPJ putative binding regions on the TFF2 promoter. (E) RT‐qPCR analysis of TFF2 binding site expression of GES‐1 cells. Target site expression in RBPJ‐treated groups was normalized to IgG negative control groups and expressed as fold change relative to the IgG groups. (F) IF staining of NICD (pink) and DAPI (blue). Scale bar = 50 µm. (G) IF analysis of MFI of nuclear NICD in positive cells. (H) Representative immunoblotting band of Notch signaling pathway in GES‐1 cells. (I–K) Analyses of immunoblotting band gray value of (H). (L) RT‐qPCR analysis of TFF2 mRNA expression of DMSO/DAPT treated GES‐1 cells. Target gene expression was normalized to GAPDH mRNA and expressed as fold change relative to the DMSO vehicle group. (M) RT‐qPCR analysis of TFF2 mRNA expression in RBPJ overexpressed and NICD deprived GES‐1 cells. The target gene expression was normalized to GAPDH mRNA and expressed as fold change relative to the vector + DMSO group. (N) RT‐qPCR analysis of TFF2 mRNA expression in RBPJ overexpressed and NICD‐RBPJ overexpressed GES‐1 cells. The target gene expression was normalized to GAPDH mRNA and expressed as fold change relative to the vector1 + vector2 group. (O) Representative image of PLA of NICD–RBPJ proximity. Each red dot represents a positive signal of NICD–RBPJ interaction and nuclei were counterstained with DAPI (blue). Scale bar = 20 µm. Quantitative data are expressed as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and ns denotes no significance. Nit, nitrate; EtOH, ethanol; GES‐1, human gastric epithelial; NICD, intracellular structural domain; RBPJ, recombination signal binding protein for immunoglobulin kappa J region; EMSA, electrophoretic mobility shift assay; TFF2, trefoil factor 2; DAPI, 2‐(4‐amidinophenyl)‐6‐indolecarbamidine dihydrochloride; Yhhu‐3792, N2‐(4‐isopropylphenyl)‐5‐(3‐methoxyphenoxy) quinazoline‐2,4‐diamine; Luc, luciferase; SD, standard deviation; DLR, dual‐luciferase report; PLA, proximity ligation assay.

    Journal: MedComm

    Article Title: Nitrate Enhances Gastric Mucosa Defense and Repair Process in Ethanol‐Induced Gastric Ulcer Rats via the Notch–Tff2 Pathway

    doi: 10.1002/mco2.70628

    Figure Lengend Snippet: Nitrate functions by Notch pathway inhibition positively transcripting TFF2 in vitro. (A) Diagram of the wild‐type and mutant sequences for the three predicted RBPJ binding sites within the 251 bp TFF2 promoter probes. (B) Representative electrophoretic mobility shift assay (EMSA) autoradiograph. Hot probe is the biotin‐labeled wild‐type oligonucleotides of the truncated TFF2 promoter containing the binding motif; Mutant probe is the labeled oligonucleotides sequence with nucleotides mutated. The cold probe is nonlabeled competitive wild‐type probes (100 and 50 that of the concentrations). The shifted bands are indicated by arrows, which suggested the formation of DNA–protein complexes (lane 2, 3, 7). The super shifted bands indicated the formation of DNA–protein–antibody complexes (lane 3, 7). “+” and “−” represent presence and absence, respectively. (C) Relative TFF2 promoter (Full, Mut1, Mut2, and Mut3) luciferase activity was detected by DLR assays in RBPJ overexpressed and normal‐expressed GES‐1 cells. (D) A schematic diagram showing the location of RBPJ putative binding regions on the TFF2 promoter. (E) RT‐qPCR analysis of TFF2 binding site expression of GES‐1 cells. Target site expression in RBPJ‐treated groups was normalized to IgG negative control groups and expressed as fold change relative to the IgG groups. (F) IF staining of NICD (pink) and DAPI (blue). Scale bar = 50 µm. (G) IF analysis of MFI of nuclear NICD in positive cells. (H) Representative immunoblotting band of Notch signaling pathway in GES‐1 cells. (I–K) Analyses of immunoblotting band gray value of (H). (L) RT‐qPCR analysis of TFF2 mRNA expression of DMSO/DAPT treated GES‐1 cells. Target gene expression was normalized to GAPDH mRNA and expressed as fold change relative to the DMSO vehicle group. (M) RT‐qPCR analysis of TFF2 mRNA expression in RBPJ overexpressed and NICD deprived GES‐1 cells. The target gene expression was normalized to GAPDH mRNA and expressed as fold change relative to the vector + DMSO group. (N) RT‐qPCR analysis of TFF2 mRNA expression in RBPJ overexpressed and NICD‐RBPJ overexpressed GES‐1 cells. The target gene expression was normalized to GAPDH mRNA and expressed as fold change relative to the vector1 + vector2 group. (O) Representative image of PLA of NICD–RBPJ proximity. Each red dot represents a positive signal of NICD–RBPJ interaction and nuclei were counterstained with DAPI (blue). Scale bar = 20 µm. Quantitative data are expressed as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and ns denotes no significance. Nit, nitrate; EtOH, ethanol; GES‐1, human gastric epithelial; NICD, intracellular structural domain; RBPJ, recombination signal binding protein for immunoglobulin kappa J region; EMSA, electrophoretic mobility shift assay; TFF2, trefoil factor 2; DAPI, 2‐(4‐amidinophenyl)‐6‐indolecarbamidine dihydrochloride; Yhhu‐3792, N2‐(4‐isopropylphenyl)‐5‐(3‐methoxyphenoxy) quinazoline‐2,4‐diamine; Luc, luciferase; SD, standard deviation; DLR, dual‐luciferase report; PLA, proximity ligation assay.

    Article Snippet: The probes were incubated with the GES‐1 cells extract and RBPJ antibody (5313; Cell Signaling Technology, USA) at room temperature for 50 min. To comprehend the specificity of the DNA/protein binding reactions, competition assays were performed with 50/100 excessive unlabeled cold probes.

    Techniques: Inhibition, In Vitro, Mutagenesis, Binding Assay, Electrophoretic Mobility Shift Assay, Autoradiography, Labeling, Sequencing, Luciferase, Activity Assay, Quantitative RT-PCR, Expressing, Negative Control, Staining, Western Blot, Targeted Gene Expression, Plasmid Preparation, Standard Deviation, Proximity Ligation Assay

    (A) Bar plots showing the frequency of LSK (left) and HSCs (LSK CD48 − CD150 + ) (right) in RBPj vav1 WT and KO E14.5 FL embryos. Each dot represents one embryo (n=10). (B) Box plots showing cell cycle analysis of RBPj vav1 WT and KO HSCs (LSK CD48 − CD150 + ) from E14.5 FL, assessed by Ki67 and DAPI staining. Each dot represents one embryo (n=10). (C) Competitive transplantation of the indicated numbers of E14.5 FL cells from RBPj vav1 WT and KO mice (CD45.2 + ) together with 2×10 5 C57BL/6 BM competitor cells (CD45.1 + ) into lethally irradiated recipient mice (CD45.1 + ). Graph shows the percentage of engraftment in BM 16 weeks after primary transplantation. Each dot represents one mouse (n=4-8). (D) Limiting dilution assay (LDA) to determine the colony-repopulating unit (CRU) frequency of E14.5 FL HSCs from RBPj vav1 WT and KO embryos, analyzed by ELDA (Extreme Limiting Dilution Analysis) at 16 weeks post-transplantation. The table summarizes the estimated HSC frequencies obtained from the LDA. N=4-8 mice. (E) UMAP representation of active, intermediate and dormant HSC populations from RBPj vav1 WT and KO E14.5 FL LSK scRNA-seq data, based on the Dormant HSC signature and the Active_HSC_MPPs signature. N=2 pooled embryos. (F) Expression of HSC-related genes (upper; Hlf, Mecom, Procr, Mllt3) and active HSC-associated genes (bottom; CD34, Myc, Cdk4, Cdk6) in HSC subcluster represented in UMAP. N=2 pooled embryos. (G-I) Box plots showing score representation of HSC Score (G); Signature scores including adult HSC dormancy, serial-engrafting FL HSCs, diapause, low-output, activated HSC/MPPs, high-output, MSigDB, Hallmark Myc target genes version 1 (V1) and version 2 (V2) (H); and Dormancy module score (I) in dormant HSC populations from RBPj vav1 WT and KO E14.5 FL LSK scRNA-seq data. N=2 pooled embryos. (J) Box plots showing cell cycle analysis of WT and NICD vav1 HSCs (LSK CD48 − CD150 + ) from E14.5 FL. Each dot represents one embryo (n=6). (K) Bar plots showing the frequency of LSK (left), HSCs (LSK CD48 − CD150 + ) (middle) and MPP3/MPP4 (LSK CD48 + CD150 − ) in WT and NICD vav1 E14.5 FL. Each dot represents one embryo (n=6). (L) Bar plot showing GSEA results from bulk RNA-seq analysis comparing NICD vav1 versus WT HSCs (LSK CD48 − CD150 + ), using gene signatures previously associated with dormant HSCs, activated HSCs/MPPs, metabolic activity, cell cycle and Notch signaling, including the Notch_signaling hallmark (MSigDB_HALLMARK) as well as signatures for High-output, serially engrafting FL HSCs and serially engrafting BM HSCs. Only statistically significant pathways are shown (adjusted p-value < 0.1). Pathways are sorted in descending order of normalized enrichment score (NES). The experiment included five samples in total (n=3 NICD vav1 HSCs and n=2 WT HSCs). Box plots: center line indicates the median; lower and upper hinges represent the first and third quartiles; whiskers extend to the largest and smallest values within 1.5× the interquartile range (IQR). No outliers are present.

    Journal: bioRxiv

    Article Title: Developmental programming of hematopoietic stem cell dormancy by evasion of Notch signaling

    doi: 10.64898/2026.01.02.697352

    Figure Lengend Snippet: (A) Bar plots showing the frequency of LSK (left) and HSCs (LSK CD48 − CD150 + ) (right) in RBPj vav1 WT and KO E14.5 FL embryos. Each dot represents one embryo (n=10). (B) Box plots showing cell cycle analysis of RBPj vav1 WT and KO HSCs (LSK CD48 − CD150 + ) from E14.5 FL, assessed by Ki67 and DAPI staining. Each dot represents one embryo (n=10). (C) Competitive transplantation of the indicated numbers of E14.5 FL cells from RBPj vav1 WT and KO mice (CD45.2 + ) together with 2×10 5 C57BL/6 BM competitor cells (CD45.1 + ) into lethally irradiated recipient mice (CD45.1 + ). Graph shows the percentage of engraftment in BM 16 weeks after primary transplantation. Each dot represents one mouse (n=4-8). (D) Limiting dilution assay (LDA) to determine the colony-repopulating unit (CRU) frequency of E14.5 FL HSCs from RBPj vav1 WT and KO embryos, analyzed by ELDA (Extreme Limiting Dilution Analysis) at 16 weeks post-transplantation. The table summarizes the estimated HSC frequencies obtained from the LDA. N=4-8 mice. (E) UMAP representation of active, intermediate and dormant HSC populations from RBPj vav1 WT and KO E14.5 FL LSK scRNA-seq data, based on the Dormant HSC signature and the Active_HSC_MPPs signature. N=2 pooled embryos. (F) Expression of HSC-related genes (upper; Hlf, Mecom, Procr, Mllt3) and active HSC-associated genes (bottom; CD34, Myc, Cdk4, Cdk6) in HSC subcluster represented in UMAP. N=2 pooled embryos. (G-I) Box plots showing score representation of HSC Score (G); Signature scores including adult HSC dormancy, serial-engrafting FL HSCs, diapause, low-output, activated HSC/MPPs, high-output, MSigDB, Hallmark Myc target genes version 1 (V1) and version 2 (V2) (H); and Dormancy module score (I) in dormant HSC populations from RBPj vav1 WT and KO E14.5 FL LSK scRNA-seq data. N=2 pooled embryos. (J) Box plots showing cell cycle analysis of WT and NICD vav1 HSCs (LSK CD48 − CD150 + ) from E14.5 FL. Each dot represents one embryo (n=6). (K) Bar plots showing the frequency of LSK (left), HSCs (LSK CD48 − CD150 + ) (middle) and MPP3/MPP4 (LSK CD48 + CD150 − ) in WT and NICD vav1 E14.5 FL. Each dot represents one embryo (n=6). (L) Bar plot showing GSEA results from bulk RNA-seq analysis comparing NICD vav1 versus WT HSCs (LSK CD48 − CD150 + ), using gene signatures previously associated with dormant HSCs, activated HSCs/MPPs, metabolic activity, cell cycle and Notch signaling, including the Notch_signaling hallmark (MSigDB_HALLMARK) as well as signatures for High-output, serially engrafting FL HSCs and serially engrafting BM HSCs. Only statistically significant pathways are shown (adjusted p-value < 0.1). Pathways are sorted in descending order of normalized enrichment score (NES). The experiment included five samples in total (n=3 NICD vav1 HSCs and n=2 WT HSCs). Box plots: center line indicates the median; lower and upper hinges represent the first and third quartiles; whiskers extend to the largest and smallest values within 1.5× the interquartile range (IQR). No outliers are present.

    Article Snippet: Mice used were C57BL/6J (Jackson Laboratories, Strain #000664), CD-1 IGS (Charles Rivers, Strain #022), Vav-Cre (B6.Cg-Tg(VAV1-cre)1Graf/MdfJ) (Jackson Laboratories, Strain #035670), RBPj flox (C57BL/6J-Rbpj em2Lutzy /J) (Jackson Laboratories, Strain # 034200), eEF1a NICD (C57BL/6J-Tg(eEF1a-NICD,-loxP-stop-loxP)) mice were kindly provided by Dr. Iannis Aifantis, ROSA26 M2rtTa/M2rtTA (B6.Cg-Gt(ROSA)26Sor tm1(rtTA*M2)Jae /J) (Jackson Laboratories, Strain #006965), pTRE-H2BGFP +/GFP (Tg(tetO-HIST1H2BJ/GFP)47Efu/J (Jackson Laboratories, Strain #005104).

    Techniques: Cell Cycle Assay, Staining, Transplantation Assay, Irradiation, Limiting Dilution Assay, Expressing, RNA Sequencing, Activity Assay