anti-notch4 monoclonal antibodies clone hmn4-14  (Bio X Cell)

Journal: Cell reports

Article Title: Akt3 activation by R-Ras in an endothelial cell enforces quiescence and barrier stability of neighboring endothelial cells via Jagged1

doi: 10.1016/j.celrep.2024.113837

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-Notch4 (A-12) , Santa Cruz , sc-393893;.

Techniques: Virus, Control, Expressing, Plasmid Preparation, Recombinant, Isolation, MTT Assay, Western Blot, Software

Journal: bioRxiv

Article Title: Breast Cancer Macrophage Heterogeneity and Self-renewal are Determined by Spatial Localization

doi: 10.1101/2023.10.24.563749

Figure Lengend Snippet: ( a ) Macrophages plated either directly with oTME cells (cell contact) or separated by transwells for 7 days, followed by 72 hrs of EdU labeling. Macrophages were imaged (mT) at plating and prior to flow cytometry analysis of EdU. Individual dots represent individual fields of view from 3 independent experiments. Data are shown as mean±SD, 2-tailed t-test. Scale bar, 100μm. ( b ) Gene Set Enrichment Analysis of Gene Ontology showing a significant enrichment of Notch signaling pathway in oTME macrophages (GO:0007219; FDR=0.00065; top, enrichment scores; bottom, rank positions of member genes). ( c ) Rosa26 mTmG mice (n=8) with established mammary tumors were treated with Notch4 monoclonal antibodies (15μg/kg body weight, dosed every 3 days, intraperitoneally) or vehicle (PBS) as a control for 20 days. Tumor sizes were recorded by a caliper and shown as mean ±SD, 2-way ANOVA test, Bonferroni-corrected. At the trial endpoint, mammary tumors were imaged (right). ( d ) Macrophage proliferation analysis in mammary tumors from ( c ) following Notch4 treatment (n=6) at study endpoint. Tumors were harvested and scored for Ki67 and CD206 expression in macrophages by flow cytometry. Data are shown as mean±SD, 2-way ANOVA test, Bonferroni-corrected. Macrophage abundance quantified as percent of live CD45+ cells. Data are shown as mean±SD, Mann-Whitney test. ( e ) C57BL/6 female mice were injected with luciferase-expressing HGSOC cells (1 x 10 6 , intraperitoneally) and treated 7 days after tumor BLI signals were established with NOTCH4 or isotype control IgG antibodies (15μg/kg body weight, dosed every 3 days, intraperitoneally). BLI measurements were recorded every week and representative images are presented.

Article Snippet: Then, mice were pooled and randomized into two arms: vehicle-treated (PBS) or Notch4-treated with anti-Notch4 monoclonal antibodies (BioXcell, clone HMN4-14).

Techniques: Labeling, Flow Cytometry, Bioprocessing, Control, Expressing, MANN-WHITNEY, Injection, Luciferase

Journal: bioRxiv

Article Title: Breast Cancer Macrophage Heterogeneity and Self-renewal are Determined by Spatial Localization

doi: 10.1101/2023.10.24.563749

Figure Lengend Snippet: ( a ) Immunoblotting for the cleaved Notch intracellular domain (NICD), ARG1, phospho-PI3K (pAKT), and phospho-MAPK (pERK), confirming active Notch and PI3K pathways in oTME macrophages but not in CSF-1 macrophages. ( b ) Impact of γ-secretase inhibition on proliferation of oTME macrophages. BM monocytes were isolated and plated with oTME cells for 7 days and then were treated with either compound-E (CompE; 10μM) or DMSO as a control for additional 10 days. Macrophages and oTME cells were analyzed by flow cytometry for Ki67 expression. Data (n=3) are shown as mean±SD, Welch’s t- test. ( c ) EdU incorporation in BMDMs following treatment with Adam17 protease inhibitor (A17Pro) or PBS as vehicle control. Cells were plated with oTME cells in the presence of A17Pro or PBS and cultured for seven days. Cell cultures were then labeled with EdU for 48hrs and were analyzed by flow cytometry for EdU incorporation (n=4 replicates). Data are shown as mean±SD, Welch’s t-test. MFI; mean fluorescence intensity. ( d ) Flow cytometry of relative abundances of Ki67+ tumor cells (mT neg CD45 neg ), total immune cells (mT+ CD45 + ), and stromal cells (mT+ CD45 neg ) in control vs. NOTCH4-treated tumors (n=6 replicates). Data are shown as mean ±SD, 2-way ANOVA test, Bonferroni-corrected. ( e ) Immunofluorescence staining of CD31, IBA1, and Ki67 in tumor transplants from (a). Scale bars, 100μm. ( f ) Primary tumor growth (left) and lung metastasis (right) of metastatic TNBC PDX cells (BR18) in NSG mice following NOTCH4 and IgG treatment. Mice were engrafted with 50K cells and treated as in ( c). Growth kinetics and lung dissemination were evaluated by bioluminescence.

Article Snippet: Then, mice were pooled and randomized into two arms: vehicle-treated (PBS) or Notch4-treated with anti-Notch4 monoclonal antibodies (BioXcell, clone HMN4-14).

Techniques: Western Blot, Inhibition, Isolation, Control, Flow Cytometry, Expressing, Protease Inhibitor, Cell Culture, Labeling, Fluorescence, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Breast Cancer Macrophage Heterogeneity and Self-renewal are Determined by Spatial Localization

doi: 10.1101/2023.10.24.563749

Figure Lengend Snippet: ( a ) S patial P r O tein and T ranscriptome S equencing (SPOTS) on tissue from MMTV-PyMT model (n=3 mice). ( b ) Tissue structure. IF staining with EpCAM-PE and CD45-APC to reveal the tissue architecture. Scale bar, 200μm. ( c ) Spatial clustering and ADT signatures of each cluster. Left panel: spatially-informed clusters (1-6) overlaid onto tissue spatial barcodes. Right panel: heatmap of ADT expression for each cluster, where the right colorbar represents the cell-type annotation of each ADT. ( d ) Spatial ADT expression levels of key surface markers for tumor (EpCAM), fibroblasts (PDPN), and macrophages (F4/80, CD86) in fibroblast-enriched region (cluster 2) and adenocarcinoma (cluster 5). Note enrichment of EpCAM high CD86 high expression in adenocarcinoma region (cluster 5; consistent with TNMs) vs. enrichment of PDPN high F4/80 high expression in fibroblasts-enriched regions (cluster 2; consistent with SAMs). ( e ) Spatial correlation of lineage (NK1.1, CD4, CD8, CD11b, F4/80, MHC-II, EpCAM) immunostimulatory (CD27, CD86, CCR2, CD11c), and immunosuppressive (PD-L1, Sca-1) ADTs in fibroblast-enriched region (cluster 2) and adenocarcinoma region (cluster 5). ADTs are colored by bivariate Moran’s I (color scale). The size of each dot represents the inverse of standard error of the mean (S.E.M; n=3 mice). Dendrograms indicate the hierarchical clustering of the ADTs. ( f ) EpCAM and CCR2 vs. PDPN and F4/80 ADTs co-expression levels overlaid onto tumor sample A tissue (Methods) and immunophenotyping of SAMs and TNMs. Middle panel: boxplots of EpCAM and CCR2 vs. PDPN and F4/80 co-expression levels (addition of EpCAM and CCR2, or PDPN and F4/80 expression values). Right panel: violin plots of immune-stimulatory and suppressive ADT expression levels. Kolmogorov–Smirnov test, * P < 0.05, ** P < 0.01, *** P < 0.001, otherwise not significant (n.s). Each boxplot ranges from the first and third quartiles with median values shown as middle lines, and the whiskers represent 1.5 times the interquartile range. ( g ) Volcano plot showing log fold changes (logFC) of top 5,000 most variable genes between fibroblast-enriched region (cluster 2) vs. adenocarcinoma region (cluster 5) and their significance (y axis; -log10 scale). Genes are dotted and colored by logFC levels (color scale). The size of each dot represents the difference in the fraction of detection between the two groups. Macrophage-related genes are annotated. P -values were determined by Wilcoxon Rank Sum test. Vertical dotted lines represent ±0.2 logFC. Horizontal dotted lines represent FDR of 0.05 (-log10 scale). Raw, FDR corrected P -values, and logFC values are listed in Supplementary Table 10. ( h ) Gene Set Enrichment Analysis of scRNA-seq Ly6a +SAM gene signature (Supplementary Table 7; ) in the fibroblast-enriched region (cluster 2) ( P = 0.002). ( i ) Gene expression of IFNα response genes ( Ly6a, Ly6c1, Ifit1, Ifit3, Ifitm3, Irf7, Isg15, Stat1, Stat2 ) in SAMs (cluster 2) and TNMs (cluster 5). Genes are dotted and colored by expression levels. The size of each dot represents the percentage of expression in the tissue area. ( j ) Notch signaling pathway activity in fibroblast-enriched regions (SAMs) and adenocarcinoma regions (TNMs) across all three biological replicates. Left panel: violin plots of NOTCH4 ADT expression levels. Right panel: violin plots of Notch signaling pathway (GO:0007219; n=179 genes) transcriptional expression levels. Kolmogorov–Smirnov test, * P < 0.05, ** P < 0.01, *** P < 0.001, otherwise not significant (n.s).

Article Snippet: Then, mice were pooled and randomized into two arms: vehicle-treated (PBS) or Notch4-treated with anti-Notch4 monoclonal antibodies (BioXcell, clone HMN4-14).

Techniques: Staining, Expressing, Gene Expression, Activity Assay

Journal: bioRxiv

Article Title: Intestinal microbiota programming of alveolar macrophages influences severity of respiratory viral infection

doi: 10.1101/2023.09.21.558814

Figure Lengend Snippet: (A) Schematic design for ex vivo studies of AM. (B) Comparison of basal SFB + and SFB - AM gene expression by RNA-seq, shown as a volcano plot. (C) SFB + and SFB - AM were exposed to UV-CA09 with or without Notch4 neutralizing, or isotype control antibodies. RNA was harvested 24 hours later, and gene expression assayed by RT-qPCR. Results are a heat map summary with full results shown in FS7. (D) AM and CA09 stocks were incubated at 37C, or indicated temperature, for 45 minutes and supernatant were collected. In addition, some cells had new media added and were incubated for another 24 hours. (i) virus infection titers in supernatant at 45 minutes and 24 hours were assayed. (ii) Supernatant were collected after 45 minutes incubated with CA09 and viral genomes quantitated by RT-qPCR. (iii) Cell-free supernatant of 15 hours AM cultures were incubated with live CA09 stocks, with or without C1qa-neutralization or isotype antibody for 45 minutes, at which point virus infection titers were assayed. (iv) Cell lysates were generated upon CA09 removal or 24 hours later and viral genomes quantitated by RT-qPCR. All experiment n = 4-5 mice per group. Data is representative of two independent experiments, yielding an identical pattern of results. Statistical analysis: One-way ANOVA. *p<0.05, **p<0.01,, ****p<0.0001, ns not significant.

Article Snippet: After sorting, cells were treated with either isotype control antibody (Cat# BE0091, BioXCell) or an anti-Notch4 monoclonal antibody (20μg/mL) (Cat# BE0129, BioXCell), known to neutralize Notch 4 [ ] for 30 minutes.

Techniques: Ex Vivo, Comparison, Gene Expression, RNA Sequencing, Control, Quantitative RT-PCR, Incubation, Virus, Infection, Neutralization, Generated

Journal: bioRxiv

Article Title: Intestinal microbiota programming of alveolar macrophages influences severity of respiratory viral infection

doi: 10.1101/2023.09.21.558814

Figure Lengend Snippet: (A) As schematized, AM was isolated from SFB - and SFB + mice and treated with UV-inactivated CA09 in the presence/absence of anti-Notch4 monoclonal antibodies. qRT-PCR results of Il6, and type I interferon-related genes from AM. (B) (i) As schematized, CD45.1 SFB - mice were administered SFB or PBS. Seven days later, AMs were isolated by FACs. Some SFB - AM were then treated with an anti-Notch4 monoclonal antibody or isotype. AM were then transferred to CD45.2 SFB - mice, 5×10 5 per mouse. One day post transfer, mice were inoculated with CA09. Mice were euthanized on day 4.5 post virus inoculation. FACs graphical representation, frequencies, and cell numbers of endogenous and exogenous AM. (ii) lung CA09 viral genomes copies, survival rate and body weight. (iii) FACs graphical representation and frequencies of AECs-containing CA09. All experiments n =5 mice per group. Data is representative of two independent experiments, yielding an identical pattern of results. Statistical test: Relative expression of pro-inflammatory genes, CA09 RNA copies, frequencies of GFP+ cells data: one-way ANOVA. Survival data; one-way ANOVA. Body weight: Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns not significant.

Article Snippet: After sorting, cells were treated with either isotype control antibody (Cat# BE0091, BioXCell) or an anti-Notch4 monoclonal antibody (20μg/mL) (Cat# BE0129, BioXCell), known to neutralize Notch 4 [ ] for 30 minutes.

Techniques: Isolation, Bioprocessing, Quantitative RT-PCR, Virus, Expressing

Journal: Burns & Trauma

Article Title: Notch4 participates in mesenchymal stem cell-induced differentiation in 3D-printed matrix and is implicated in eccrine sweat gland morphogenesis

doi: 10.1093/burnst/tkad032

Figure Lengend Snippet: Primers of genes for real-time quantitative polymerase chain reaction

Article Snippet: Furthermore, to specifically inhibit Notch4 receptors without disturbing other Notch signaling-related molecules, anti-Notch4 monoclonal antibody (5 mg/kg per mouse, sc-393 893, Santa Cruz) was intraperitoneally injected into two pregnant mice once daily for 2 days [ ].

Techniques: Marker

Journal: Burns & Trauma

Article Title: Notch4 participates in mesenchymal stem cell-induced differentiation in 3D-printed matrix and is implicated in eccrine sweat gland morphogenesis

doi: 10.1093/burnst/tkad032

Figure Lengend Snippet: Notch signals regulate SG-originated cell stemness and are implicated in embryonic SG morphogenesis in vivo . Schematic drawings of DAPT injection and checking points, i.e. E12.5, E17.5, P1, P5 and P14 ( a ). Immunohistochemistry staining of Notch4 ( b) , Oct4 ( c) and Krt19 ( d) . Immunofluorescence staining of Notch4 and Krt14 ( e ). Red arrows (b): Notch4-expressing positions. Black arrows (b–d) and white arrows (e): secretory coils of SG. Red dotted box (b–e): two critical time points during SG morphogenesis (i.e. E17.5 and P14). Scale bar (b): 200 μm, 50 μm; (c–e): 50 μm. DAPT N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-(S)-phenylglycine t -butyl, E embryonic day, Krt keratin, P postpartum day, SG sweat gland

Article Snippet: Furthermore, to specifically inhibit Notch4 receptors without disturbing other Notch signaling-related molecules, anti-Notch4 monoclonal antibody (5 mg/kg per mouse, sc-393 893, Santa Cruz) was intraperitoneally injected into two pregnant mice once daily for 2 days [ ].

Techniques: In Vivo, Injection, Immunohistochemistry, Staining, Immunofluorescence, Expressing

Journal: Burns & Trauma

Article Title: Notch4 participates in mesenchymal stem cell-induced differentiation in 3D-printed matrix and is implicated in eccrine sweat gland morphogenesis

doi: 10.1093/burnst/tkad032

Figure Lengend Snippet: Notch4 specifically targets Krt19-positive epidermal stem cells and Krt14-positive SG progenitor cells in vivo . Schematic drawings of anti-Notch4 monoclonal antibodies injection and checking points, i.e. E17.5 and P14 ( a ). Immunohistochemistry staining of Notch4 ( b ), Oct4 ( c ) and Krt19 ( d ). Immunofluorescence staining of Notch4 (red color) and Krt14 (green color) ( e ). Flow chart of this study ( f ). Red arrows (b): Notch4-expressing positions. Scale bar (b): 200 μm, 50 μm; (e): 50 μm. DAPT N -[ N -(3,5-Difluorophenacetyl-L-alanyl)]-(S)-phenylglycine t -butyl, E embryonic day, GO Gene Ontology, iSG induced sweat gland, Krt keratin, MSCs mesenchymal stem cells, P postpartum day, SG sweat gland, VPA valproic acid

Article Snippet: Furthermore, to specifically inhibit Notch4 receptors without disturbing other Notch signaling-related molecules, anti-Notch4 monoclonal antibody (5 mg/kg per mouse, sc-393 893, Santa Cruz) was intraperitoneally injected into two pregnant mice once daily for 2 days [ ].

Techniques: In Vivo, Bioprocessing, Injection, Immunohistochemistry, Staining, Immunofluorescence, Expressing

Journal: Burns & Trauma

Article Title: Notch4 participates in mesenchymal stem cell-induced differentiation in 3D-printed matrix and is implicated in eccrine sweat gland morphogenesis

doi: 10.1093/burnst/tkad032

Figure Lengend Snippet: Notch4 is one of the niche-dual-responding genes and is related to MSC stemness in the 3D matrix in vitro . Pairwise comparisons of ( a ) ‘3D MSC vs 2D MSC’, ( b ) ‘3D iSG vs 2D MSC’ and ( c ) ‘3D iSG vs 3D MSC’ revealed differentially expressed genes. ( d ) GO analysis showed these differentially expressed genes were enriched in cell stemness- (green font) and angiogenesis- (yellow font) related GO terms. Overlapping filtered out 76 genes and the heat map shows their transcriptional profiles. The dotted box indicates a cluster of structural cue-responsive genes. ( e ) The intersection of these 76 genes with ‘GO:0035239 tube morphogenesis’-enriched genes resulted in six candidate genes, i.e. Hmox1, Notch4, Cited1, Pdgfb, E2f8 and Mylk. GO Gene Ontology, iSG induced sweat gland, MAPK mitogen-activated protein kinase, MSCs mesenchymal stem cells

Article Snippet: Furthermore, to specifically inhibit Notch4 receptors without disturbing other Notch signaling-related molecules, anti-Notch4 monoclonal antibody (5 mg/kg per mouse, sc-393 893, Santa Cruz) was intraperitoneally injected into two pregnant mice once daily for 2 days [ ].

Techniques: In Vitro

Journal: Burns & Trauma

Article Title: Notch4 participates in mesenchymal stem cell-induced differentiation in 3D-printed matrix and is implicated in eccrine sweat gland morphogenesis

doi: 10.1093/burnst/tkad032

Figure Lengend Snippet: Notch4 specifically regulated Oct4-related stemness of MSCs in 3D matrix in vitro . ( a ) Inhibiting Notch signaling slowed down the proliferation rate of MSCs. Immunofluorescence staining of Notch4 and Oct4 in 2D culture system ( b ) and 3D culture system ( c ). ( d ) Western blotting images of Notch4 and Oct4 in response to administration of DAPT or VPA. ( e ) Relative density quantifications of Notch4 and Oct4 protein levels; n = 3. Gene expression of Notch4 and Oct4 exhibit a similar profile in both 2D culture system ( f ) (n = 3) and 3D culture system ( g ) (n = 3). Gene expressions of Notch1, 2, 3 and cell stemness markers (Nanog and Sox2) in 2D culture system ( h ) (n = 3) and 3D culture system ( i ) (n = 3). Green dotted lines indicate gene expression levels of ‘2D MSC’ group (f–i). Scale bar (a): 200 μm; (b, c): 50 μm. DAPT N -[ N -(3,5-Difluorophenacetyl-L-alanyl)]-(S)-phenylglycine t -butyl, DMSO dimethyl sulfoxide, KDa kilodalton, MSCs mesenchymal stem cells, NS no significant difference, RT-qPCR real-time quantitative polymerase chain reaction, VPA valproic acid

Article Snippet: Furthermore, to specifically inhibit Notch4 receptors without disturbing other Notch signaling-related molecules, anti-Notch4 monoclonal antibody (5 mg/kg per mouse, sc-393 893, Santa Cruz) was intraperitoneally injected into two pregnant mice once daily for 2 days [ ].

Techniques: In Vitro, Immunofluorescence, Staining, Western Blot, Gene Expression, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: Burns & Trauma

Article Title: Notch4 participates in mesenchymal stem cell-induced differentiation in 3D-printed matrix and is implicated in eccrine sweat gland morphogenesis

doi: 10.1093/burnst/tkad032

Figure Lengend Snippet: Notch4 mediates SG differentiation of MSCs in the 3D model in vitro . Immunofluorescence staining of Notch4 (red color) and Krt18 (green color) in 2D ( a ) and 3D ( b ) culture system. Western blotting of Notch4 and Krt18 in 2D and 3D culture system ( c ). Gene expression of Notch4 ( d ), Krt18 ( e ), Krt14 ( f ) and ATP1a1 ( g ); n = 3. Scale bar (a): 50 μm; (b): 20 μm. ATP1a1 ATPase Na+/K+ transporting subunit alpha 1, DAPT N -[ N -(3,5-difluorophenacetyl-L-alanyl)]-(S)-phenylglycine t -butyl, DMSO dimethyl sulfoxide, KDa kilodalton, Krt keratin, MSCs mesenchymal stem cells, NS no significant difference, PBS phosphate-buffered saline, RT-qPCR real-time quantitative polymerase chain reaction, VPA valproic acid

Article Snippet: Furthermore, to specifically inhibit Notch4 receptors without disturbing other Notch signaling-related molecules, anti-Notch4 monoclonal antibody (5 mg/kg per mouse, sc-393 893, Santa Cruz) was intraperitoneally injected into two pregnant mice once daily for 2 days [ ].

Techniques: In Vitro, Immunofluorescence, Staining, Western Blot, Gene Expression, Saline, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: Burns & Trauma

Article Title: Notch4 participates in mesenchymal stem cell-induced differentiation in 3D-printed matrix and is implicated in eccrine sweat gland morphogenesis

doi: 10.1093/burnst/tkad032

Figure Lengend Snippet: Primers of genes for real-time quantitative polymerase chain reaction

Article Snippet: For detecting the relationships between Notch4 and Oct4, the slides were incubated with anti-Notch4 mouse monoclonal antibody (1 : 100, sc-393 893, Santa Cruz) and anti-Oct4 rabbit polyclonal antibody (1 : 250, ab18976, Abcam).

Techniques: Marker

Journal: Burns & Trauma

Article Title: Notch4 participates in mesenchymal stem cell-induced differentiation in 3D-printed matrix and is implicated in eccrine sweat gland morphogenesis

doi: 10.1093/burnst/tkad032

Figure Lengend Snippet: Notch signals regulate SG-originated cell stemness and are implicated in embryonic SG morphogenesis in vivo . Schematic drawings of DAPT injection and checking points, i.e. E12.5, E17.5, P1, P5 and P14 ( a ). Immunohistochemistry staining of Notch4 ( b) , Oct4 ( c) and Krt19 ( d) . Immunofluorescence staining of Notch4 and Krt14 ( e ). Red arrows (b): Notch4-expressing positions. Black arrows (b–d) and white arrows (e): secretory coils of SG. Red dotted box (b–e): two critical time points during SG morphogenesis (i.e. E17.5 and P14). Scale bar (b): 200 μm, 50 μm; (c–e): 50 μm. DAPT N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-(S)-phenylglycine t -butyl, E embryonic day, Krt keratin, P postpartum day, SG sweat gland

Article Snippet: For detecting the relationships between Notch4 and Oct4, the slides were incubated with anti-Notch4 mouse monoclonal antibody (1 : 100, sc-393 893, Santa Cruz) and anti-Oct4 rabbit polyclonal antibody (1 : 250, ab18976, Abcam).

Techniques: In Vivo, Injection, Immunohistochemistry, Staining, Immunofluorescence, Expressing

Journal: Burns & Trauma

Article Title: Notch4 participates in mesenchymal stem cell-induced differentiation in 3D-printed matrix and is implicated in eccrine sweat gland morphogenesis

doi: 10.1093/burnst/tkad032

Figure Lengend Snippet: Notch4 specifically targets Krt19-positive epidermal stem cells and Krt14-positive SG progenitor cells in vivo . Schematic drawings of anti-Notch4 monoclonal antibodies injection and checking points, i.e. E17.5 and P14 ( a ). Immunohistochemistry staining of Notch4 ( b ), Oct4 ( c ) and Krt19 ( d ). Immunofluorescence staining of Notch4 (red color) and Krt14 (green color) ( e ). Flow chart of this study ( f ). Red arrows (b): Notch4-expressing positions. Scale bar (b): 200 μm, 50 μm; (e): 50 μm. DAPT N -[ N -(3,5-Difluorophenacetyl-L-alanyl)]-(S)-phenylglycine t -butyl, E embryonic day, GO Gene Ontology, iSG induced sweat gland, Krt keratin, MSCs mesenchymal stem cells, P postpartum day, SG sweat gland, VPA valproic acid

Article Snippet: For detecting the relationships between Notch4 and Oct4, the slides were incubated with anti-Notch4 mouse monoclonal antibody (1 : 100, sc-393 893, Santa Cruz) and anti-Oct4 rabbit polyclonal antibody (1 : 250, ab18976, Abcam).

Techniques: In Vivo, Bioprocessing, Injection, Immunohistochemistry, Staining, Immunofluorescence, Expressing

Journal: Burns & Trauma

Article Title: Notch4 participates in mesenchymal stem cell-induced differentiation in 3D-printed matrix and is implicated in eccrine sweat gland morphogenesis

doi: 10.1093/burnst/tkad032

Figure Lengend Snippet: Notch4 is one of the niche-dual-responding genes and is related to MSC stemness in the 3D matrix in vitro . Pairwise comparisons of ( a ) ‘3D MSC vs 2D MSC’, ( b ) ‘3D iSG vs 2D MSC’ and ( c ) ‘3D iSG vs 3D MSC’ revealed differentially expressed genes. ( d ) GO analysis showed these differentially expressed genes were enriched in cell stemness- (green font) and angiogenesis- (yellow font) related GO terms. Overlapping filtered out 76 genes and the heat map shows their transcriptional profiles. The dotted box indicates a cluster of structural cue-responsive genes. ( e ) The intersection of these 76 genes with ‘GO:0035239 tube morphogenesis’-enriched genes resulted in six candidate genes, i.e. Hmox1, Notch4, Cited1, Pdgfb, E2f8 and Mylk. GO Gene Ontology, iSG induced sweat gland, MAPK mitogen-activated protein kinase, MSCs mesenchymal stem cells

Article Snippet: For detecting the relationships between Notch4 and Oct4, the slides were incubated with anti-Notch4 mouse monoclonal antibody (1 : 100, sc-393 893, Santa Cruz) and anti-Oct4 rabbit polyclonal antibody (1 : 250, ab18976, Abcam).

Techniques: In Vitro

Journal: Burns & Trauma

Article Title: Notch4 participates in mesenchymal stem cell-induced differentiation in 3D-printed matrix and is implicated in eccrine sweat gland morphogenesis

doi: 10.1093/burnst/tkad032

Figure Lengend Snippet: Notch4 specifically regulated Oct4-related stemness of MSCs in 3D matrix in vitro . ( a ) Inhibiting Notch signaling slowed down the proliferation rate of MSCs. Immunofluorescence staining of Notch4 and Oct4 in 2D culture system ( b ) and 3D culture system ( c ). ( d ) Western blotting images of Notch4 and Oct4 in response to administration of DAPT or VPA. ( e ) Relative density quantifications of Notch4 and Oct4 protein levels; n = 3. Gene expression of Notch4 and Oct4 exhibit a similar profile in both 2D culture system ( f ) (n = 3) and 3D culture system ( g ) (n = 3). Gene expressions of Notch1, 2, 3 and cell stemness markers (Nanog and Sox2) in 2D culture system ( h ) (n = 3) and 3D culture system ( i ) (n = 3). Green dotted lines indicate gene expression levels of ‘2D MSC’ group (f–i). Scale bar (a): 200 μm; (b, c): 50 μm. DAPT N -[ N -(3,5-Difluorophenacetyl-L-alanyl)]-(S)-phenylglycine t -butyl, DMSO dimethyl sulfoxide, KDa kilodalton, MSCs mesenchymal stem cells, NS no significant difference, RT-qPCR real-time quantitative polymerase chain reaction, VPA valproic acid

Article Snippet: For detecting the relationships between Notch4 and Oct4, the slides were incubated with anti-Notch4 mouse monoclonal antibody (1 : 100, sc-393 893, Santa Cruz) and anti-Oct4 rabbit polyclonal antibody (1 : 250, ab18976, Abcam).

Techniques: In Vitro, Immunofluorescence, Staining, Western Blot, Gene Expression, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: Burns & Trauma

Article Title: Notch4 participates in mesenchymal stem cell-induced differentiation in 3D-printed matrix and is implicated in eccrine sweat gland morphogenesis

doi: 10.1093/burnst/tkad032

Figure Lengend Snippet: Notch4 mediates SG differentiation of MSCs in the 3D model in vitro . Immunofluorescence staining of Notch4 (red color) and Krt18 (green color) in 2D ( a ) and 3D ( b ) culture system. Western blotting of Notch4 and Krt18 in 2D and 3D culture system ( c ). Gene expression of Notch4 ( d ), Krt18 ( e ), Krt14 ( f ) and ATP1a1 ( g ); n = 3. Scale bar (a): 50 μm; (b): 20 μm. ATP1a1 ATPase Na+/K+ transporting subunit alpha 1, DAPT N -[ N -(3,5-difluorophenacetyl-L-alanyl)]-(S)-phenylglycine t -butyl, DMSO dimethyl sulfoxide, KDa kilodalton, Krt keratin, MSCs mesenchymal stem cells, NS no significant difference, PBS phosphate-buffered saline, RT-qPCR real-time quantitative polymerase chain reaction, VPA valproic acid

Article Snippet: For detecting the relationships between Notch4 and Oct4, the slides were incubated with anti-Notch4 mouse monoclonal antibody (1 : 100, sc-393 893, Santa Cruz) and anti-Oct4 rabbit polyclonal antibody (1 : 250, ab18976, Abcam).

Techniques: In Vitro, Immunofluorescence, Staining, Western Blot, Gene Expression, Saline, Quantitative RT-PCR, Real-time Polymerase Chain Reaction