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Cell Signaling Technology Inc antibodies against runx1
Antibodies Against Runx1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/antibodies against runx1/product/Cell Signaling Technology Inc
Average 94 stars, based on 33 article reviews

antibodies against runx1 - by Bioz Stars, 2026-03

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Fig. 4 PRMT1 inhibition promotes proliferation and migration of ADSCs through RUNX1-mediated MMP-10/PlGF/TGF-β2 expression in ADSCs. (A) Pre dicted transcription factors binding to the target Mmp10, Plgf and Tgfb2 genes promoter region. The promoter region is defined as 2000 bp upstream of the target gene transcription starting point. (B) Predict possible binding sites in the gene promoter region and transcription factor RUNX1. (C, D) The relationship between PRMT1 and RUNX1 was detected by Co-IP experiment. (E, F) ADSCs were treated with Furamidine (10 µM) and Ro5-3335 (50 µM) for 48 h. Full-length blots are presented in Supplementary Fig. 4E, F. Western blotting and quantitative analysis of MMP-10, PlGF, and TGF-β2 protein levels (n = 6). Full-length blots are presented in Supplementary Fig. 5A. (G) Changes in cell proliferation were detected by CCK-8 after ADSCs were treated with Furamidine and Ro5-3335 for 24 h (n = 6). (H, I) Representative images of crystal violet staining. The migration ability of ADSCs was observed by transwell assay after treatment with Furamidine and Ro5-3335 (n = 4). Scale bar: 50 μm. All data are presented as mean ± SEM and were analyzed by one-way ANOVA, *P < 0.05

Journal: Stem cell research & therapy

Article Title: PRMT1 inhibition enhances the cardioprotective effect of adipose-derived mesenchymal stem cells against myocardial infarction through RUNX1.

doi: 10.1186/s13287-025-04409-z

Figure Lengend Snippet: Fig. 4 PRMT1 inhibition promotes proliferation and migration of ADSCs through RUNX1-mediated MMP-10/PlGF/TGF-β2 expression in ADSCs. (A) Pre dicted transcription factors binding to the target Mmp10, Plgf and Tgfb2 genes promoter region. The promoter region is defined as 2000 bp upstream of the target gene transcription starting point. (B) Predict possible binding sites in the gene promoter region and transcription factor RUNX1. (C, D) The relationship between PRMT1 and RUNX1 was detected by Co-IP experiment. (E, F) ADSCs were treated with Furamidine (10 µM) and Ro5-3335 (50 µM) for 48 h. Full-length blots are presented in Supplementary Fig. 4E, F. Western blotting and quantitative analysis of MMP-10, PlGF, and TGF-β2 protein levels (n = 6). Full-length blots are presented in Supplementary Fig. 5A. (G) Changes in cell proliferation were detected by CCK-8 after ADSCs were treated with Furamidine and Ro5-3335 for 24 h (n = 6). (H, I) Representative images of crystal violet staining. The migration ability of ADSCs was observed by transwell assay after treatment with Furamidine and Ro5-3335 (n = 4). Scale bar: 50 μm. All data are presented as mean ± SEM and were analyzed by one-way ANOVA, *P < 0.05

Article Snippet: The primary antibodies used in this study included: anti-PRMT1 rabbit monoclonal antibody (2449 S, 1:1000; Cell Signaling Technology, MA, USA), anti-cleaved caspase-3 rabbit polyclonal antibody (9664 S, 1:1000; Cell Signaling Technology, MA, USA), anti-MMP-10 rabbit polyclonal antibody (A3033, 1:1000; Abclonal Technology, Wuhan, China), anti-TGF-β2 rabbit polyclonal antibody (19999-1-AP, 1:1000; Proteintech, Wuhan, China), anti-PlGF rabbit polyclonal antibody (10642-1-AP, 1:1000; Proteintech, Wuhan, China), anti-RUNX1 rabbit polyclonal antibody (25315-1-AP, 1:1000; Proteintech, Wuhan, China), anti-GAPDH rabbit polyclonal antibody (10494-1-AP, 1:10000; Proteintech, Wuhan, China).

Techniques: Inhibition, Migration, Expressing, Binding Assay, Co-Immunoprecipitation Assay, Western Blot, CCK-8 Assay, Staining, Transwell Assay

Fig. 8 Schematic illustration of inhibition of PRMT1 improve the therapeutic efficacy of ADSCs for MI. After MI, the level of PRMT1 in ADSCs transplanted into the infarct border zone increased under stress, and by interacting with RUNX1, the transcription-promoting effect of RUNX1 on Mmp10, Plgf and Tgfb2 was inhibited, resulting in impaired retention and survival of ADSCs, and inadequate cardioprotective effects (left). After knockdown of PRMT1, the released RUNX1 promoted the expression of Mmp10, Plgf and Tgfb2 in ADSCs, improved the retention rate of implanted ADSCs, and enhanced cardiopro tective effects of ADSCs (right). The Figure was created with BioRender software (https://biorender.com/)

Journal: Stem cell research & therapy

Article Title: PRMT1 inhibition enhances the cardioprotective effect of adipose-derived mesenchymal stem cells against myocardial infarction through RUNX1.

doi: 10.1186/s13287-025-04409-z

Figure Lengend Snippet: Fig. 8 Schematic illustration of inhibition of PRMT1 improve the therapeutic efficacy of ADSCs for MI. After MI, the level of PRMT1 in ADSCs transplanted into the infarct border zone increased under stress, and by interacting with RUNX1, the transcription-promoting effect of RUNX1 on Mmp10, Plgf and Tgfb2 was inhibited, resulting in impaired retention and survival of ADSCs, and inadequate cardioprotective effects (left). After knockdown of PRMT1, the released RUNX1 promoted the expression of Mmp10, Plgf and Tgfb2 in ADSCs, improved the retention rate of implanted ADSCs, and enhanced cardiopro tective effects of ADSCs (right). The Figure was created with BioRender software (https://biorender.com/)

Techniques: Inhibition, Drug discovery, Knockdown, Expressing, Software

A. Time to tumour onset of PyMT+ mice wild type ( n = 44; WT ) or deleted for Runx1 ( n = 34; R1KO ). Average time to tumour onset with standard deviation shown: 61 days (± 11.5) for WT; 50 days (± 10.9) for R1KO mice. Each data point represents an individual mouse. NP: nulliparous. Statistical analysis performed with two-tailed unpaired t-test, *** P = 0.0001. B. Flow cytometric analysis of tdRFP expression in MMECs from PyMT+/WT ( n = 6) and PyMT+/R1KO ( n = 6) mice at tumour notice, and PyMT -negative controls ( PyMT-/WT, n = 7; PyMT-/R1KO, n = 6) at ∼60 days. Each data point represents percentage of tdRFP -positive MMECs, with smaller points representing individual glands (5-7 glands per mouse, see methods) and larger points representing the means from each mouse. Glands isolated from the same mouse are indicated by the same colour. Statistical analysis performed on the mean percentage of tdRFP -positive MMECs with ordinary one-way ANOVA with Sidak’s multiple comparisons test, ** P < 0.01. C. Time to tumour onset of BLG-Cre;Ctnnb1 wt/lox(ex3) ( B-cat+ ) mice wild type ( n = 12; WT ) or deleted for Runx1 ( n = 16; parous R1KO and n = 14; nulliparous R1KO ). Average time to tumour onset with standard deviation shown: 344 days (± 69.6) for parous WT ; 250 days (± 59.3) for parous R1KO ; 259 days (± 75.9) for nulliparous R1KO mice. Each data point represents an individual mouse. Parous (P) mice, filled circles; nulliparous (NP) mice, open circles. Statistical analysis performed with ordinary one-way ANOVA test with Dunnett’s multiple comparisons test, ** P < 0.01. D. Time to tumour onset in BLG-Cre;Trp53 fl/fl ( Trp53-/- ) parous (P) and nulliparous (NP) mice wild type ( n = 9 and n = 13, respectively; WT ) and deleted for Runx1 ( n = 8 and n = 15, respectively; R1KO ). Each data point represents an individual mouse. Average time to tumour onset with standard deviation shown. WT -P: 199 days (± 29.4); WT -NP: 195 days (± 26.7); R1KO- P: 195 days (± 15); R1KO -NP: 215 days (± 28.9). Statistical analysis performed with ordinary one-way ANOVA test with Sidak’s multiple comparisons test; ns, non-significant ( P > 0.05).

Journal: bioRxiv

Article Title: Runx1 and Runx2 act in concert to suppress Wnt/β-catenin -driven mammary tumourigenesis

doi: 10.1101/2025.03.16.643517

Figure Lengend Snippet: A. Time to tumour onset of PyMT+ mice wild type ( n = 44; WT ) or deleted for Runx1 ( n = 34; R1KO ). Average time to tumour onset with standard deviation shown: 61 days (± 11.5) for WT; 50 days (± 10.9) for R1KO mice. Each data point represents an individual mouse. NP: nulliparous. Statistical analysis performed with two-tailed unpaired t-test, *** P = 0.0001. B. Flow cytometric analysis of tdRFP expression in MMECs from PyMT+/WT ( n = 6) and PyMT+/R1KO ( n = 6) mice at tumour notice, and PyMT -negative controls ( PyMT-/WT, n = 7; PyMT-/R1KO, n = 6) at ∼60 days. Each data point represents percentage of tdRFP -positive MMECs, with smaller points representing individual glands (5-7 glands per mouse, see methods) and larger points representing the means from each mouse. Glands isolated from the same mouse are indicated by the same colour. Statistical analysis performed on the mean percentage of tdRFP -positive MMECs with ordinary one-way ANOVA with Sidak’s multiple comparisons test, ** P < 0.01. C. Time to tumour onset of BLG-Cre;Ctnnb1 wt/lox(ex3) ( B-cat+ ) mice wild type ( n = 12; WT ) or deleted for Runx1 ( n = 16; parous R1KO and n = 14; nulliparous R1KO ). Average time to tumour onset with standard deviation shown: 344 days (± 69.6) for parous WT ; 250 days (± 59.3) for parous R1KO ; 259 days (± 75.9) for nulliparous R1KO mice. Each data point represents an individual mouse. Parous (P) mice, filled circles; nulliparous (NP) mice, open circles. Statistical analysis performed with ordinary one-way ANOVA test with Dunnett’s multiple comparisons test, ** P < 0.01. D. Time to tumour onset in BLG-Cre;Trp53 fl/fl ( Trp53-/- ) parous (P) and nulliparous (NP) mice wild type ( n = 9 and n = 13, respectively; WT ) and deleted for Runx1 ( n = 8 and n = 15, respectively; R1KO ). Each data point represents an individual mouse. Average time to tumour onset with standard deviation shown. WT -P: 199 days (± 29.4); WT -NP: 195 days (± 26.7); R1KO- P: 195 days (± 15); R1KO -NP: 215 days (± 28.9). Statistical analysis performed with ordinary one-way ANOVA test with Sidak’s multiple comparisons test; ns, non-significant ( P > 0.05).

Article Snippet: The following antibodies were used: RUNX1 [#8529, D4A6, Cell Signaling; 1:1000]; RUNX2 [#8486, D1H7, Cell Signaling; 1:1000]; GAPDH [#3683, 14C10, Cell Signaling; 1:1000] and horseradish peroxidase- conjugated anti-rabbit secondary antibody [#7074, Cell Signaling; 1:10 000].

Techniques: Standard Deviation, Two Tailed Test, Expressing, Isolation

A. Time to tumour onset in B-cat+ mice wild-type for Runx ( n = 12; WT as in ), Runx1 fl/fl ( n = 16; R1KO as in ), Runx2 fl/fl ( n = 15; R2KO ) and Runx1 fl/fl ;Runx2 fl/fl ( n = 32; DKO ). Parous (P) mice, solid lines; nulliparous (NP) mice, dotted lines. Median survival of tumour-free mice with standard deviation shown: WT -P mice, 330 days (± 69.6); R1KO -P mice, 266 days (± 59.3); R2KO -P mice, 367 days (± 65.4); DKO -NP mice, 56 days (± 14.9). Statistical analysis performed with log-rank (Mantel-Cox) test, *** P < 0.001; **** P < 0.0001. B. Number of tumour-bearing mammary glands in B-cat+ cohorts from harvested at clinical endpoint. Each data point represents an individual mouse. Parous (P) mice, filled circles; nulliparous (NP) mice, open circles. WT -P ( n = 9); R1KO -P ( n = 12); R2KO -P ( n = 12); DKO -NP ( n = 24). Statistical analysis performed with ordinary one- way ANOVA with Dunnett’s multiple comparisons test, * P < 0.05; **** P < 0.0001. C. Wholemounts and H&E images of abdominal mammary glands from 6- and 9-weeks old B-cat+ cohorts. One representative wholemount and H&E image is shown per genotype (of n = 5-7 mice per genotype). Scale bars of wholemounts = 2mm; H&E images = 1mm. Arrow illustrates lymph node.

Journal: bioRxiv

Article Title: Runx1 and Runx2 act in concert to suppress Wnt/β-catenin -driven mammary tumourigenesis

doi: 10.1101/2025.03.16.643517

Figure Lengend Snippet: A. Time to tumour onset in B-cat+ mice wild-type for Runx ( n = 12; WT as in ), Runx1 fl/fl ( n = 16; R1KO as in ), Runx2 fl/fl ( n = 15; R2KO ) and Runx1 fl/fl ;Runx2 fl/fl ( n = 32; DKO ). Parous (P) mice, solid lines; nulliparous (NP) mice, dotted lines. Median survival of tumour-free mice with standard deviation shown: WT -P mice, 330 days (± 69.6); R1KO -P mice, 266 days (± 59.3); R2KO -P mice, 367 days (± 65.4); DKO -NP mice, 56 days (± 14.9). Statistical analysis performed with log-rank (Mantel-Cox) test, *** P < 0.001; **** P < 0.0001. B. Number of tumour-bearing mammary glands in B-cat+ cohorts from harvested at clinical endpoint. Each data point represents an individual mouse. Parous (P) mice, filled circles; nulliparous (NP) mice, open circles. WT -P ( n = 9); R1KO -P ( n = 12); R2KO -P ( n = 12); DKO -NP ( n = 24). Statistical analysis performed with ordinary one- way ANOVA with Dunnett’s multiple comparisons test, * P < 0.05; **** P < 0.0001. C. Wholemounts and H&E images of abdominal mammary glands from 6- and 9-weeks old B-cat+ cohorts. One representative wholemount and H&E image is shown per genotype (of n = 5-7 mice per genotype). Scale bars of wholemounts = 2mm; H&E images = 1mm. Arrow illustrates lymph node.

Techniques: Standard Deviation

A. Graph (top) and representative pictures (bottom) of colony-forming cell assay showing relative proportions of acinar- and solid-like colonies from MMECs isolated from 7-9 weeks old B-cat + cohorts; Runx1 wild type ( n = 5; WT ); R1KO ( n = 4); DKO ( n = 3). Mean with standard error of n ≥ 3 independent experiments, each with 12 technical replicates per condition. One representative image shown per cohort ( n ≥ 3 biological replicates per cohort). Acinar and solid-like colonies depicted by white and black arrows, respectively. Statistical analysis performed with two-way ANOVA with Dunnett’s multiple comparisons test, **** P < 0.0001. Scale bar, 500µm. B. Analysis of mammary RNA-seq signatures derived from gene set enrichment analysis (GSEA) of FACS-sorted tdRFP- positive MMECs from 9-weeks old B-cat + females comparing three conditions: Runx1 knockout ( R1KO; n = 5) versus wildtype for Runx genes ( WT; n = 4), Runx1/Runx2 knockout ( DKO; n = 5) versus WT , and DKO versus R1KO . The gene sets analysed include LIM Mammary Stem Cell Up ( https://www.gsea/msigdb.org/gsea-msigdb/mouse/geneset/LIM_MAMMARY_STEM_CELL_UP.html ), LIM Mammary Mature Luminal Up ( https://www.gsea-msigdb.org/gsea/msigdb/mouse/geneset/LIM_MAMMARY_STEM_CELL_UP.html ), and LIM Mammary Luminal Progenitor Up ( https://www.gsea-msigdb.org/gsea/msigdb/mouse/geneset/LIM_MAMMARY_STEM_CELL_UP.html ). Each bar represents the normalised enrichment score (NES) for a specific gene set under each comparison, with higher NES values indicating stronger enrichment of the gene set in the corresponding condition. C. Normalised gene expression levels of selected genes in FACS-sorted tdRFP- positive MMECs as described for . Individual data points represent expression levels per sample, with central line indicating the median. Gene expression normalised for sequencing depth, with significant differences between conditions assessed with Wald test and shown as p-values adjusted for multiple testing with Benjamini-Hochberg method. Both procedures performed with DESeq2; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Journal: bioRxiv

Article Title: Runx1 and Runx2 act in concert to suppress Wnt/β-catenin -driven mammary tumourigenesis

doi: 10.1101/2025.03.16.643517

Figure Lengend Snippet: A. Graph (top) and representative pictures (bottom) of colony-forming cell assay showing relative proportions of acinar- and solid-like colonies from MMECs isolated from 7-9 weeks old B-cat + cohorts; Runx1 wild type ( n = 5; WT ); R1KO ( n = 4); DKO ( n = 3). Mean with standard error of n ≥ 3 independent experiments, each with 12 technical replicates per condition. One representative image shown per cohort ( n ≥ 3 biological replicates per cohort). Acinar and solid-like colonies depicted by white and black arrows, respectively. Statistical analysis performed with two-way ANOVA with Dunnett’s multiple comparisons test, **** P < 0.0001. Scale bar, 500µm. B. Analysis of mammary RNA-seq signatures derived from gene set enrichment analysis (GSEA) of FACS-sorted tdRFP- positive MMECs from 9-weeks old B-cat + females comparing three conditions: Runx1 knockout ( R1KO; n = 5) versus wildtype for Runx genes ( WT; n = 4), Runx1/Runx2 knockout ( DKO; n = 5) versus WT , and DKO versus R1KO . The gene sets analysed include LIM Mammary Stem Cell Up ( https://www.gsea/msigdb.org/gsea-msigdb/mouse/geneset/LIM_MAMMARY_STEM_CELL_UP.html ), LIM Mammary Mature Luminal Up ( https://www.gsea-msigdb.org/gsea/msigdb/mouse/geneset/LIM_MAMMARY_STEM_CELL_UP.html ), and LIM Mammary Luminal Progenitor Up ( https://www.gsea-msigdb.org/gsea/msigdb/mouse/geneset/LIM_MAMMARY_STEM_CELL_UP.html ). Each bar represents the normalised enrichment score (NES) for a specific gene set under each comparison, with higher NES values indicating stronger enrichment of the gene set in the corresponding condition. C. Normalised gene expression levels of selected genes in FACS-sorted tdRFP- positive MMECs as described for . Individual data points represent expression levels per sample, with central line indicating the median. Gene expression normalised for sequencing depth, with significant differences between conditions assessed with Wald test and shown as p-values adjusted for multiple testing with Benjamini-Hochberg method. Both procedures performed with DESeq2; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Techniques: Isolation, RNA Sequencing, Derivative Assay, Knock-Out, Comparison, Gene Expression, Expressing, Sequencing

A. Scatter dot-plot of primary (left) and secondary (right) mammospheres of HC11 cells with empty vector (Empty) or independent CRISPR/Cas9-deleted Runx1 clones (R1KO_a, R1KO_b, R1KO_c). Results show normalised mammosphere counts per well relative to the average mammosphere count in control (Empty) group. Smaller points represent technical replicates, larger points the mean of each experimental repeat with coloured points used to differentiate experimental replicates (also for B-D below). For primary mammospheres, there were 12-48 technical replicates per condition per experimental replicate ( n = 8). Error bars indicate normalised mean of all technical replicates ± standard deviation (Empty, 100 ± 11.5; R1KO_a , 158.1 ± 17.9; R1KO_b , 165.4 ± 20.7; R1KO_c , 166.3 ± 20.0). For secondary mammospheres, there were 12-24 technical replicates per condition for each experimental repeat ( n = 4). Error bars indicate normalised mean of all technical replicates ± standard deviation (Empty, 100.1 ± 9.6; R1KO_a , 179.1 ± 28.9; R1KO_b, 175.6 ± 31.1; R1KO_c , 174.5 ± 34.1). Statistical analysis performed with ordinary one-way ANOVA with Dunnett’s multiple comparisons test, **** P < 0.0001. B. Scatter dot-plot of primary mammospheres of HC11 cells overexpressing Runx1 ( Runx1P1 ) or empty vector control (Empty). Results show mammosphere counts relative to the average mammosphere count in control group (Empty). Error bars indicate normalised mean of all technical replicates ± standard deviation (Empty, 100 ± 18.6; Runx1P1 , 73.5 ±14.3) and data are representative of n = 4 experimental repeats. For each experiment, there were 8-12 technical replicates per condition. Statistical analysis performed with two-tailed unpaired t-test, **** P < 0.0001. C. Scatter dot-plot of primary mammospheres of HC11 cells first deleted for Runx2 and then transduced with lentiCRISPRv2-Neo empty vector (Empty) or containing a Runx1 -targeting gRNA plasmid to generate an independent Runx1 -deleted ( R1KO_d ), two Runx2 -deleted ( R2KO_a , R2KO_b ) and two Runx1/Runx2 -deleted ( DKO_a, DKO_b ) clones. Results show normalised mammosphere counts per well relative to the average mammosphere count in control group (empty). Error bars are normalised mean of all technical replicates with standard deviation (Empty, 100 ± 7.4 ; R1KO_d, 137.1 ± 9.1; R2KO_a , 80.1 ± 7.6; R2KO_b , 78.6 ± 9.6; DKO_a, 120.6 ± 7.9; DKO_b, 116.4 ± 7.7) and data are representative of n = 3 experimental repeats. For each experiment, there were 12 technical replicates per condition. Statistical analysis performed with ordinary one-way ANOVA with Dunnett’s multiple comparisons test, **** P < 0.0001. D. Scatter dot-plot of primary mammospheres of HC11 cells as for C in the presence or absence of recombinant WNT3A ligand. Error bars show mean of all technical replicates ± standard deviation for vehicle treated (Empty, 120.5 ± 6.2; R1KO_d , 145.4 ± 7.0; DKO_a , 136.8 ± 7.7; DKO_b , 137.7 ± 7.9) and WNT3A treated (Empty, 152.5 ± 8.1; R1KO_d , 191.3 ± 9.0; DKO_a , 193.4 ± 10.2; DKO_b, 193.1 ± 9.5) clones, of n = 2 experimental repeats. For each experiment, 12 technical replicates were tested per condition. Statistical analysis performed with ordinary one-way ANOVA with Sidak’s multiple comparisons test, **** P < 0.0001.

Journal: bioRxiv

Article Title: Runx1 and Runx2 act in concert to suppress Wnt/β-catenin -driven mammary tumourigenesis

doi: 10.1101/2025.03.16.643517

Figure Lengend Snippet: A. Scatter dot-plot of primary (left) and secondary (right) mammospheres of HC11 cells with empty vector (Empty) or independent CRISPR/Cas9-deleted Runx1 clones (R1KO_a, R1KO_b, R1KO_c). Results show normalised mammosphere counts per well relative to the average mammosphere count in control (Empty) group. Smaller points represent technical replicates, larger points the mean of each experimental repeat with coloured points used to differentiate experimental replicates (also for B-D below). For primary mammospheres, there were 12-48 technical replicates per condition per experimental replicate ( n = 8). Error bars indicate normalised mean of all technical replicates ± standard deviation (Empty, 100 ± 11.5; R1KO_a , 158.1 ± 17.9; R1KO_b , 165.4 ± 20.7; R1KO_c , 166.3 ± 20.0). For secondary mammospheres, there were 12-24 technical replicates per condition for each experimental repeat ( n = 4). Error bars indicate normalised mean of all technical replicates ± standard deviation (Empty, 100.1 ± 9.6; R1KO_a , 179.1 ± 28.9; R1KO_b, 175.6 ± 31.1; R1KO_c , 174.5 ± 34.1). Statistical analysis performed with ordinary one-way ANOVA with Dunnett’s multiple comparisons test, **** P < 0.0001. B. Scatter dot-plot of primary mammospheres of HC11 cells overexpressing Runx1 ( Runx1P1 ) or empty vector control (Empty). Results show mammosphere counts relative to the average mammosphere count in control group (Empty). Error bars indicate normalised mean of all technical replicates ± standard deviation (Empty, 100 ± 18.6; Runx1P1 , 73.5 ±14.3) and data are representative of n = 4 experimental repeats. For each experiment, there were 8-12 technical replicates per condition. Statistical analysis performed with two-tailed unpaired t-test, **** P < 0.0001. C. Scatter dot-plot of primary mammospheres of HC11 cells first deleted for Runx2 and then transduced with lentiCRISPRv2-Neo empty vector (Empty) or containing a Runx1 -targeting gRNA plasmid to generate an independent Runx1 -deleted ( R1KO_d ), two Runx2 -deleted ( R2KO_a , R2KO_b ) and two Runx1/Runx2 -deleted ( DKO_a, DKO_b ) clones. Results show normalised mammosphere counts per well relative to the average mammosphere count in control group (empty). Error bars are normalised mean of all technical replicates with standard deviation (Empty, 100 ± 7.4 ; R1KO_d, 137.1 ± 9.1; R2KO_a , 80.1 ± 7.6; R2KO_b , 78.6 ± 9.6; DKO_a, 120.6 ± 7.9; DKO_b, 116.4 ± 7.7) and data are representative of n = 3 experimental repeats. For each experiment, there were 12 technical replicates per condition. Statistical analysis performed with ordinary one-way ANOVA with Dunnett’s multiple comparisons test, **** P < 0.0001. D. Scatter dot-plot of primary mammospheres of HC11 cells as for C in the presence or absence of recombinant WNT3A ligand. Error bars show mean of all technical replicates ± standard deviation for vehicle treated (Empty, 120.5 ± 6.2; R1KO_d , 145.4 ± 7.0; DKO_a , 136.8 ± 7.7; DKO_b , 137.7 ± 7.9) and WNT3A treated (Empty, 152.5 ± 8.1; R1KO_d , 191.3 ± 9.0; DKO_a , 193.4 ± 10.2; DKO_b, 193.1 ± 9.5) clones, of n = 2 experimental repeats. For each experiment, 12 technical replicates were tested per condition. Statistical analysis performed with ordinary one-way ANOVA with Sidak’s multiple comparisons test, **** P < 0.0001.

Techniques: Plasmid Preparation, CRISPR, Clone Assay, Control, Standard Deviation, Two Tailed Test, Transduction, Recombinant

$A. PCA plot of the top 500 most variable genes showing RNA-seq expression profiles for whole mouse mammary glands harvested from 9-weeks old B-cat + mice. Each point represents an individual mouse either: Runx wildtype (WT; black; n = 5), R1KO (blue; n = 5), R2KO (green; n = 5), and DKO (red; n = 4). PC1 and PC2, explaining 55% and 16% of the variance, are on the x- and y-axes, respectively. B. Top enriched pathways from MetaCore’s Canonical Pathway Maps representing signalling and metabolic pathways are shown (for samples as in A). The -log10 of the FDR-corrected p-value indicates the probability of the observed intersection between the DEGs and the pathway gene set occurring by chance, as determined by a hypergeometric test. A higher -log10 p-value suggests a stronger association between the DEGs and the pathway “DKO vs WT” refers to DEGs in the Runx1 / Runx2 double knockout compared to wildtype. C. Heatmap displaying z-scored mMCP scores for immune and stromal cell populations in Runx wildtype (WT; n = 5) versus Runx1 / Runx2 double knockout (DKO; n = 4) 9- weeks old B-cat+ mammary glands (as in A). Each column represents an individual mouse. D. Heatmap of CIBERSORTx immune cell fractions in Runx wildtype (WT; n = 5) versus Runx1 / Runx2 double knockout (DKO; n = 4) 9-weeks old B-cat+ mammary glands (as in C and D). CIBERSORTx immune cell fractions calculated using a signature matrix created from single cell sequencing of BLG-cre;Brca1 f/f ;Trp53 +/- murine mammary glands from Bach et al . . Z-scores are representative of the gene expression relative to the mean expression, indicated by a diverging scale running from blue to red. Each column represents an individual mouse.$

Journal: bioRxiv

Article Title: Runx1 and Runx2 act in concert to suppress Wnt/β-catenin -driven mammary tumourigenesis

doi: 10.1101/2025.03.16.643517

Figure Lengend Snippet: A. PCA plot of the top 500 most variable genes showing RNA-seq expression profiles for whole mouse mammary glands harvested from 9-weeks old B-cat + mice. Each point represents an individual mouse either: Runx wildtype (WT; black; n = 5), R1KO (blue; n = 5), R2KO (green; n = 5), and DKO (red; n = 4). PC1 and PC2, explaining 55% and 16% of the variance, are on the x- and y-axes, respectively. B. Top enriched pathways from MetaCore’s Canonical Pathway Maps representing signalling and metabolic pathways are shown (for samples as in A). The -log10 of the FDR-corrected p-value indicates the probability of the observed intersection between the DEGs and the pathway gene set occurring by chance, as determined by a hypergeometric test. A higher -log10 p-value suggests a stronger association between the DEGs and the pathway “DKO vs WT” refers to DEGs in the Runx1 / Runx2 double knockout compared to wildtype. C. Heatmap displaying z-scored mMCP scores for immune and stromal cell populations in Runx wildtype (WT; n = 5) versus Runx1 / Runx2 double knockout (DKO; n = 4) 9- weeks old B-cat+ mammary glands (as in A). Each column represents an individual mouse. D. Heatmap of CIBERSORTx immune cell fractions in Runx wildtype (WT; n = 5) versus Runx1 / Runx2 double knockout (DKO; n = 4) 9-weeks old B-cat+ mammary glands (as in C and D). CIBERSORTx immune cell fractions calculated using a signature matrix created from single cell sequencing of BLG-cre;Brca1 f/f ;Trp53 +/- murine mammary glands from Bach et al . . Z-scores are representative of the gene expression relative to the mean expression, indicated by a diverging scale running from blue to red. Each column represents an individual mouse.

Techniques: RNA Sequencing, Expressing, Double Knockout, Sequencing, Gene Expression

Activation of RUNX1‐CMYB‐AQP4 signaling is downregulated in NOTCH3‐R170C astrocyte. (A) Dual‐luciferase reporter assays showing the effect of CMYB activation on Aqp4 transcription in HEK293T cells. Experiments were repeated for three times. * p < 0.05; by Student's t test (mean ± SEM). (B) Total or nucleus protein of NOTCH3‐R170C and ‐WT astrocytes were subjected to western blot. Protein level was normalized to BACT or LAMIN B1 expression. Experiments were repeated for four times. * p < 0.05, ** p < 0.01, and *** p < 0.001; by Student's t test (mean ± SEM). (C) Protein was extracted from NOTCH3‐R170C or ‐WT astrocyte then subjected to immunoprecipitation (IP) with anti‐NOTCH3 or anti‐CMYB antibodies. IP products were analyzed with western blot to examine the interaction of NOTCH3 and RUNX1 or RUNX1 and CMYB, respectively. Experiments were repeated for 3 times. * p < 0.05; by Student's t test (mean ± SEM). (D) Immunostaining of CMYB (green) and RUNX1 (red) with NOTCH3‐R170C or ‐WT astrocyte. (E, F) NOTCH3‐R170C or ‐WT OSCs were labeled with RUNX1 (green) and S100B (red) (E) or CMYB (green) and S100B (red) (F), respectively. Percentage of RUNX1 + or CMYB + cells among S100B + astrocytes was calculated. Experiments were repeated for three times. * p < 0.05, ** p < 0.01; by Student's t test (mean ± SEM). (G–H) Coronal brain sections of NOTCH3‐R170C or ‐WT mice (16 weeks of age) were subjected to immunostaining of RUNX1 (red) and S100B (green) (G) or CMYB (red) and S100B (green) (H). N = 6 in each group. * p < 0.05, ** p < 0.01; by Student's t test (mean ± SEM). (I) RUNX1 was overexpressed in NOTCH3‐R170C or ‐WT astrocytes with lentiviral vectors carrying Runx1 cDNA ( Runx1 ‐OE) which were then subjected to western blot. Protein level was normalized to GAPDH expression. Experiments were repeated for four times. * p < 0.05, ** p < 0.01; by one‐way ANOVA (mean ± SEM).

Journal: CNS Neuroscience & Therapeutics

Article Title: NOTCH3 Mutation Causes Glymphatic Impairment and Promotes Brain Senescence in CADASIL

doi: 10.1111/cns.70140

Figure Lengend Snippet: Activation of RUNX1‐CMYB‐AQP4 signaling is downregulated in NOTCH3‐R170C astrocyte. (A) Dual‐luciferase reporter assays showing the effect of CMYB activation on Aqp4 transcription in HEK293T cells. Experiments were repeated for three times. * p < 0.05; by Student's t test (mean ± SEM). (B) Total or nucleus protein of NOTCH3‐R170C and ‐WT astrocytes were subjected to western blot. Protein level was normalized to BACT or LAMIN B1 expression. Experiments were repeated for four times. * p < 0.05, ** p < 0.01, and *** p < 0.001; by Student's t test (mean ± SEM). (C) Protein was extracted from NOTCH3‐R170C or ‐WT astrocyte then subjected to immunoprecipitation (IP) with anti‐NOTCH3 or anti‐CMYB antibodies. IP products were analyzed with western blot to examine the interaction of NOTCH3 and RUNX1 or RUNX1 and CMYB, respectively. Experiments were repeated for 3 times. * p < 0.05; by Student's t test (mean ± SEM). (D) Immunostaining of CMYB (green) and RUNX1 (red) with NOTCH3‐R170C or ‐WT astrocyte. (E, F) NOTCH3‐R170C or ‐WT OSCs were labeled with RUNX1 (green) and S100B (red) (E) or CMYB (green) and S100B (red) (F), respectively. Percentage of RUNX1 + or CMYB + cells among S100B + astrocytes was calculated. Experiments were repeated for three times. * p < 0.05, ** p < 0.01; by Student's t test (mean ± SEM). (G–H) Coronal brain sections of NOTCH3‐R170C or ‐WT mice (16 weeks of age) were subjected to immunostaining of RUNX1 (red) and S100B (green) (G) or CMYB (red) and S100B (green) (H). N = 6 in each group. * p < 0.05, ** p < 0.01; by Student's t test (mean ± SEM). (I) RUNX1 was overexpressed in NOTCH3‐R170C or ‐WT astrocytes with lentiviral vectors carrying Runx1 cDNA ( Runx1 ‐OE) which were then subjected to western blot. Protein level was normalized to GAPDH expression. Experiments were repeated for four times. * p < 0.05, ** p < 0.01; by one‐way ANOVA (mean ± SEM).

Article Snippet: The following primary antibodies were used: mouse anti‐N3ECD (Sigma‐Aldrich, clone 1E4, 1:300), rabbit anti‐αSMA (Proteintech 14,395‐1‐AP, 1:100), rat anti‐CD31 (Bioscience 550,274, 1:50), rabbit anti‐AQP4 (Santa Cruz Biotechnology sc‐20,812, 1:500), mouse anti‐GFAP (CST 3670S, 1:500), rabbit anti‐RUNX1 (Affinity Biosciences AF6379, 1:300), rabbit anti‐CMYB (Affinity Biosciences AF6136, 1:300), mouse anti‐S100β (Sigma‐Aldrich S2532, 1:500), rabbit anti‐iba1 (Wako 019‐19,741,1:500), rabbit anti‐NEUN (Abcam ab177487, 1:500), mouse anti‐CC1 (Calbiochem OP80, 1:500), rabbit anti‐CDKN2A/p16INK4a (Abcam ab211542, 1:500), and rabbit anti‐p21(Affinity Biosciences AF6290, 1:300).

Techniques: Activation Assay, Luciferase, Western Blot, Expressing, Immunoprecipitation, Immunostaining, Labeling

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