anti cxcl12 sdf 1  (R&D Systems)

Journal: Cell reports

Article Title: Post-stroke hippocampal neurogenesis is impaired by microvascular dysfunction and PI3K signaling in cerebral amyloid angiopathy

doi: 10.1016/j.celrep.2024.114848

Figure Lengend Snippet: (A) Brain hippocampal tissue WT MCAO ( n = 3 mice) and 5xFAD MCAO mice ( n = 2 mice) at 12 months of age and 7 days post MCAO were subjected to scRNA-seq. Diagram showing an overview of the scRNA-seq experiment. The contralateral hippocampus was used as an internal sham control for all analyses. (B) UMAP visualization of 18,186 isolated cells at 0.2 resolution from 12-month-old mice ( n = 2 or 3 mice per group). Similar clusters are merged to show the annotated cell types within the same cluster. AC, astrocytes; LC, leukocytes; MC, myeloid cells; Neu, neurons; NPC, neural progenitor cells; OL, oligodendrocytes; PC, pericytes; PVM, perivascular macrophages. (C) UMAP plots with each cell cluster highlighted in purple corresponding to a ubiquitous marker used to identify that cluster. (D) Heatmap revealing the scaled expression of DEGs in ECs from WT MCAO and 5xFAD MCAO mice. (E) Volcano plot depicting the top upregulated and downregulated genes in EC population in WT MCAO compared to 5xFAD MCAO. Genes that are significant at a p value ≤0.05 and log fold change ≥1.25 are portrayed in red. (F) GO pathway analysis for upregulated (yellow) or downregulated (purple) genes in the EC cluster of 5xFAD MCAO mice. Bars show −log10( p ). (G) Violin plots showing the mean and variance differences between WT MCAO and 5xFAD MCAO ECs for genes regulating blood vessel morphogenesis ( Sox18, ID1 ) regulation of Neu death ( C1QA, TYROBP, CD200 ), and chemokine signaling ( Cxcl12, Ccl4 ).

Article Snippet: Primary antibodies and dilutions were applied the next day as follows: anti-ionized calcium-binding adapter molecule 1 (Iba1; 1:1000; Fisher Scientific), neuronal nuclei (NeuN; 1:500 Abcam), anti-glial fibrillary protein (GFAP; 1:1000; 3670 Cell Signaling), Lycopersicon Esculentum (Tomato) Lectin (LEL, TL), DyLight 488 (1:200; Vector Laboratories) anti-Claudin-5 (claudin-5; 1:500 Cell Signaling), anti-Occludin (Occln; 1:500 Cell Signaling), anti-zonula occludens-1 (ZO-1; 1:500 Fisher Scientific), anti-doublecortin (DCX; 1:200 Santa Cruz), anti-BrdU (1:200, Abcam), and anti-CXCL12/SDF-1 (Cxcl12; 1:500 RD Systems).

Techniques: Control, Isolation, Marker, Expressing

Journal: Cell reports

Article Title: Post-stroke hippocampal neurogenesis is impaired by microvascular dysfunction and PI3K signaling in cerebral amyloid angiopathy

doi: 10.1016/j.celrep.2024.114848

Figure Lengend Snippet: (A) Volcano plot illustrating the downregulated genes in the NPC cluster from 5xFAD MCAO mice compared to 5xFAD sham mice. Genes significant at a p value <0.05 and log fold change ≥1.25 are portrayed in red. (B) Violin plots showing the mean and variance differences between 5xFAD MCAO and 5xFAD sham NPCs for genes regulating Neu projection guidance ( Cxcl12, Scn1b ), PI3K/AKT signaling pathway ( Pik3c2a, Creb3l2 ), and RNA processing ( Ddx23, Kin ). (C) GO pathway analyses for downregulated genes in the NPC population of the 5xFAD MCAO mice. Bars show −log10( p ). (D) Brain sections from 5xFAD sham or 5xFAD MCAO mice (sham, n = 3 mice; MCAO, n = 3 mice) at 12 months of age were subjected to co-immunostaining for Cxcl12 (red) and CD31 (green), or DCX (blue), NeuN (green), and DAPI (cyan). (E) The ratio of Cxcl12/CD31 signal was quantified. ** p = 0.0011, two-tailed Student’s t test. Scale bar, 50 μm. (F) The relative fluorescent count signal of DCX per DAPI+ cell was quantified from brain sections of mice. * p = 0.0223. (G) Incucyte ZOOM analysis of NPC neurogenesis over 16 h. ReN cells were exposed to siRNA-CXCL12, siRNA-PIK3C2A, siRNA-CREB3L2, or scrambled siRNA for 24 h before imaging in the incucyte. Representative images from time points 0 and 16 h after siRNA transfection were taken, where blue indicates neurite extensions and green represents cell bodies. Scale bar, 100 μm. (H) Quantification of Incucyte images demonstrates the change in rate of neurite length per cell-body cluster area. Graphs represent the average of individual pictures taken in 16 image grids over 16 h. Additionally, t = 0 h corresponds to the first image taken after a 24-h transfection with siRNA on the graph. xy plot is mean ± SEM. ** p = 0.0058, *** p = 0.0007, **** p < 0.0001; ††† p = 0.0001, †††† p < 0.0001; ‡‡‡‡ p < 0.0001; #### p < 0.0001; ordinary two-way ANOVA with multiple comparisons.

Article Snippet: Primary antibodies and dilutions were applied the next day as follows: anti-ionized calcium-binding adapter molecule 1 (Iba1; 1:1000; Fisher Scientific), neuronal nuclei (NeuN; 1:500 Abcam), anti-glial fibrillary protein (GFAP; 1:1000; 3670 Cell Signaling), Lycopersicon Esculentum (Tomato) Lectin (LEL, TL), DyLight 488 (1:200; Vector Laboratories) anti-Claudin-5 (claudin-5; 1:500 Cell Signaling), anti-Occludin (Occln; 1:500 Cell Signaling), anti-zonula occludens-1 (ZO-1; 1:500 Fisher Scientific), anti-doublecortin (DCX; 1:200 Santa Cruz), anti-BrdU (1:200, Abcam), and anti-CXCL12/SDF-1 (Cxcl12; 1:500 RD Systems).

Techniques: Immunostaining, Two Tailed Test, Imaging, Transfection

Journal: Cell reports

Article Title: Post-stroke hippocampal neurogenesis is impaired by microvascular dysfunction and PI3K signaling in cerebral amyloid angiopathy

doi: 10.1016/j.celrep.2024.114848

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Primary antibodies and dilutions were applied the next day as follows: anti-ionized calcium-binding adapter molecule 1 (Iba1; 1:1000; Fisher Scientific), neuronal nuclei (NeuN; 1:500 Abcam), anti-glial fibrillary protein (GFAP; 1:1000; 3670 Cell Signaling), Lycopersicon Esculentum (Tomato) Lectin (LEL, TL), DyLight 488 (1:200; Vector Laboratories) anti-Claudin-5 (claudin-5; 1:500 Cell Signaling), anti-Occludin (Occln; 1:500 Cell Signaling), anti-zonula occludens-1 (ZO-1; 1:500 Fisher Scientific), anti-doublecortin (DCX; 1:200 Santa Cruz), anti-BrdU (1:200, Abcam), and anti-CXCL12/SDF-1 (Cxcl12; 1:500 RD Systems).

Techniques: Plasmid Preparation, Recombinant, Marker, Control, Saline, Lysis, Protease Inhibitor, Stripping Membranes, Purification, Bicinchoninic Acid Protein Assay, RNAscope, Multiplex Assay, Negative Control, Software, Staining, Microscopy

Journal: Scientific Reports

Article Title: SDF-1 involvement in orthodontic tooth movement after tooth extraction

doi: 10.1038/s41598-024-55632-2

Figure Lengend Snippet: Immunofluorescence (IF) staining of stromal cell-derived factor 1 (SDF-1) using Alexa Fluor® 594 (red) and DAPI (blue) for nuclear staining at the compression side of maxillary second molar (M2) mesial root on days 1,3, and 7. Positively stained cells were detected abundantly on day 3, whereas less were observed on days 1 and 7. Following neutralization via anti-SDF-1 neutralizing monoclonal antibody injection, reduced positive signals could be detected on day 3 of the SDF-1 antibody group compared to the two control groups ( a ). Number of positively stained cells at the compression side of M2 mesial root on days 1, 3, and 7. Statistically significant differences can be seen on day 1 between the SDF-1 antibody group and the PBS control group, and on day 3 between the SDF-1 antibody group and the two control groups ( b ). Scale bars = 50 μm. Abbreviations: B; buccal, M; mesial, Pa; palatal, D; distal. Values are presented as means ± standard deviation ( n = 3). * p < 0.05.

Article Snippet: After the coil spring placement, the experimental group was given a local injection of anti-SDF-1 neutralizing monoclonal antibody (MAB310; R&D Systems, Minneapolis, MN, USA) at a volume and concentration of 10 μg/0.1 mL.

Techniques: Immunofluorescence, Staining, Derivative Assay, Neutralization, Injection, Control, Standard Deviation

Journal: Science Advances

Article Title: Stromal-derived MAOB promotes prostate cancer growth and progression

doi: 10.1126/sciadv.adi4935

Figure Lengend Snippet: ( A ) GSEA plots of “chemokine signaling pathway” and “chemotaxis” gene sets enriched in MAOB-OE PrSC cells compared with controls. ( B ) ELISA of CXCL12 secretion in the culture media of control and MAOB-KD PrSC cells ( n = 3). ( C ) Representative IHC images and corresponding Pearson’s correlation analysis of stroma-expressed MAOB and CXCL12 protein levels in a PC TMA ( n = 37). Scale bars, 100 μm. ( D ) Pearson’s correlation analysis of MAOB and CXCL12 mRNA levels in patient-derived cultured prostatic stromal cells (left, n = 20) and laser-capture microdissected breast tumor stroma (right, n = 53) from GSE34312 and GSE9014 datasets, respectively. Statistical analysis was performed using one-way ANOVA with Dunnett’s test in (B). Data represent means ± SEM. ** P < 0.01.

Article Snippet: For determining the effect of stromal-derived MAOB/CXCL12-CXCR4 paracrine signaling on tumor cell proliferation in cocultures, selegiline, anti-CXCL12-neutralizing antibody (R&D Systems, MAB310, RRID:AB_2276927), rCXCL12 protein, or AMD3100 was added to cocultures 24 hours after tumor cell seeding, followed by determination of tumor cell proliferation.

Techniques: Chemotaxis Assay, Enzyme-linked Immunosorbent Assay, Control, Derivative Assay, Cell Culture

Journal: Science Advances

Article Title: Stromal-derived MAOB promotes prostate cancer growth and progression

doi: 10.1126/sciadv.adi4935

Figure Lengend Snippet: ( A ) Western blot of Twist1 in control and MAOB-manipulated PrSC cells upon NAC (5 mM, 48 hours) or H 2 O 2 (40 μM, 24 hours) treatment. ( B ) ELISA of CXCL12 secretion in culture media of control and MAOB-OE PrSC cells treated with TWIST1 siRNA or NAC (5 mM, 48 hours) ( n = 3). ( C ) qPCR of CXCL12 in indicated PrSC cells upon NAC treatment (5 mM, 48 hours) or Twist1/ TWIST1 siRNA expression ( n = 3). ( D and E ) Determination of CXCL12 mRNA (D) and 0.7-kb promoter activity (E) in PrSC cells upon Twist1 expression and/or TGFβ1 treatment (10 ng/ml, 12 hours) ( n = 3). ( F ) Schematic diagrams of WT and mutated CXCL12 E-box/SBE-Luc constructs and determination of their activities in PrSC cells upon Twist1 expression and/or TGFβ1 treatment (10 ng/ml, 12 hours) ( n = 3). ( G ) Representative proximity ligation assay staining and quantitation of indicated Twist1-Smad interactions by per-nucleus fluorescence intensity in control and MAOB-OE PrSC cells. Smad antibody incubation alone served as negative control. Numbers of nuclei included for comparisons between groups are denoted. Scale bars, 50 μm. ( H ) Co-IP assays of indicated Twist1-Smad interactions in PrSC cells with coexpression of Twist1 and individual Smads. Immunoglobulin G (IgG) was used in IP as negative control. Ten percent input was blotted as positive control. ( I ) ChIP analysis of chromatin from control and MAOB-OE PrSC cells precipitated with anti-Twist1, anti-Smad4, or a control IgG, followed by qPCR probing the E-box/SBE-centric CXCL12 promoter region ( n = 3). ( J ) ChIP analysis of chromatin from PrSC cells precipitated with anti-Smad4 antibody and then reprecipitated with anti-Twist1 or a control IgG (re-ChIP), followed by qPCR probing the E-box/SBE-encompassing CXCL12 promoter sequence ( n = 3). Statistical analysis was performed using one-way ANOVA with Tukey’s test. Data represent means ± SEM. * P < 0.05, ** P < 0.01; ns, not significant.

Article Snippet: For determining the effect of stromal-derived MAOB/CXCL12-CXCR4 paracrine signaling on tumor cell proliferation in cocultures, selegiline, anti-CXCL12-neutralizing antibody (R&D Systems, MAB310, RRID:AB_2276927), rCXCL12 protein, or AMD3100 was added to cocultures 24 hours after tumor cell seeding, followed by determination of tumor cell proliferation.

Techniques: Western Blot, Control, Enzyme-linked Immunosorbent Assay, Expressing, Activity Assay, Construct, Proximity Ligation Assay, Staining, Quantitation Assay, Fluorescence, Incubation, Negative Control, Co-Immunoprecipitation Assay, Positive Control, Sequencing

Journal: Science Advances

Article Title: Stromal-derived MAOB promotes prostate cancer growth and progression

doi: 10.1126/sciadv.adi4935

Figure Lengend Snippet: ( A ) Quantitation of C4-2 and PC-3 PC cells in monoculture and coculture with indicated PrSC fibroblasts upon anti-CXCL12 (0.1 μg/ml) antibody treatment ( n = 3). ( B ) Quantitation of PC cells in coculture with indicated PrSC cells treated with rCXCL12 protein (50 ng/ml; n = 3). ( C ) Western blot of CXCR4 and CXCR7 in control and CXCR4-KD/CXCR7-KD PC cells. ( D and E ) Quantitation of control and CXCR4-KD/CXCR7-KD PC cells in coculture with indicated PrSC cells treated without (D) or with (E) rCXCL12 (50 ng/ml; n = 3). ( F ) Quantitation of PC cells in coculture with indicated PrSC cells treated with 10 nM AMD3100 ( n = 3). ( G ) Representative images and quantitation of PC-3 cell migration and invasion in coculture with indicated PrSC cells upon treatment with anti-CXCL12 antibody (0.1 μg/ml) or 10 nM AMD3100 ( n = 3). Scale bars, 200 μm. ( H ) Representative images and quantitation of PC cell migration in coculture with indicated PrSC cells treated with rCXCL12 (50 ng/ml; n = 3). Scale bars, 100 μm. ( I ) Phospho-antibody array analysis of PC-3 cells treated with indicated PrSC cell CM. All phosphoprotein levels were normalized to their total forms from a single array with six replicate spots, with significantly activated phosphoproteins (fold change > 1.5, P < 0.05) denoted. ( J ) Western blot of p-Src and p-JNK in control and CXCR4-KD PC-3 cells treated with indicated PrSC cell CM. ( K ) Western blot of p-Src and p-JNK in PC-3 cells treated with indicated PrSC cell CM plus rCXCL12 (50 ng/ml). ( L ) Quantitation of PC cells in coculture with indicated PrSC cells following pretreatment with 40 nM Src inhibitor 1 or 10 μM SP600125 for 24 hours ( n = 3). Statistical analysis was performed using one-way ANOVA with Tukey’s test. Data represent means ± SEM. * P < 0.05, ** P < 0.01; ns, not significant.

Article Snippet: For determining the effect of stromal-derived MAOB/CXCL12-CXCR4 paracrine signaling on tumor cell proliferation in cocultures, selegiline, anti-CXCL12-neutralizing antibody (R&D Systems, MAB310, RRID:AB_2276927), rCXCL12 protein, or AMD3100 was added to cocultures 24 hours after tumor cell seeding, followed by determination of tumor cell proliferation.

Techniques: Quantitation Assay, Western Blot, Control, Migration, Ab Array

Journal: Science Advances

Article Title: Stromal-derived MAOB promotes prostate cancer growth and progression

doi: 10.1126/sciadv.adi4935

Figure Lengend Snippet: ( A ) Quantitation of C4-2 and PC-3 cell proliferation in monoculture and coculture with control and MAOB-OE PrSC cells upon selegiline treatment (10 nM, 72 hours) ( n = 3). ( B ) BLI-based growth curves of Luc-tagged PC-3 tumors grown in the prostates of male NSG mice treated with selegiline at various doses (0.5, 2, and 10 mg/kg) or saline as a vehicle ( n = 5 per group). ( C ) BLI images of mice from each group at the end point. ( D ) Determination of tumor weights ( n = 5). ( E ) Determination of MAOA and MAOB enzymatic activities in mouse liver tissue from each group at the end point ( n = 3). ( F ) Representative images of H&E and IHC staining of tumor-expressed Ki-67, p-Src, and p-JNK and stroma-expressed αSMA and CXCL12 and their quantitation in tumor samples from each group ( n = 5). Scale bars, 100 μm. ( G ) Mouse body weights determined weekly ( n = 5). ( H ) Representative H&E images of mouse liver and kidney tissue from each group. Scale bars, 100 μm. ( I to L ) ELISA of ALT (I), AST (J), BUN (K), and creatinine (L) in mouse sera at the end point ( n = 5). ( M ) Schematic depicting stromal-derived MAOB activation of paracrine CXCL12-CXCR4/Src/JNK signaling through interplay between ROS-dependent Twist1 (via a HIF1α/VEGF-A/AKT/FOXO1 pathway) and TGFβ1/Smads to promote stromal-epithelial interactions for PC growth and progression. Statistical analysis was performed using one-way ANOVA with Tukey’s test in (A), (D) to (F), and (I) to (L) and two-way ANOVA with Tukey’s test in (B) and (G). Data represent means ± SEM. * P < 0.05, ** P < 0.01; ns, not significant.

Article Snippet: For determining the effect of stromal-derived MAOB/CXCL12-CXCR4 paracrine signaling on tumor cell proliferation in cocultures, selegiline, anti-CXCL12-neutralizing antibody (R&D Systems, MAB310, RRID:AB_2276927), rCXCL12 protein, or AMD3100 was added to cocultures 24 hours after tumor cell seeding, followed by determination of tumor cell proliferation.

Techniques: Quantitation Assay, Control, Saline, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Derivative Assay, Activation Assay