Journal: Nature Communications

Article Title: CREB1-driven CXCR4 hi neutrophils promote skin inflammation in mouse models and human patients

doi: 10.1038/s41467-023-41484-3

Figure Lengend Snippet: a The mean fluorescence intensity (MFI) of CXCR4 in peripheral neutrophils from healthy controls (n = 24) and psoriasis patients (n = 54). b Correlation of the CXCR4 MFI on peripheral neutrophils with PASI in psoriasis patients (n = 25). c The proportion of circulating CD15 + CXCR4 hi neutrophils in healthy controls (n = 20) and psoriasis patients (n = 25). d Correlation of the proportions of CXCR4 hi neutrophils with PASI in psoriasis patients. The adjusted R 2 and P -values were plotted in the graph. e Representative immunoblots of total CXCR4 in circulating neutrophils from healthy controls and psoriasis patients. The relative multiple expression was counted. Blots for each antigen were processed in the same experiment in parallel. The result was repeated twice independently with similar results. f Immunofluorescence staining of CD15 (green) and CXCR4 (red) in normal and inflamed psoriatic skin. Scale bar = 50 µm. n = 10 biologically independent samples. g The proportion of CXCR4 lo vs . CXCR4 hi neutrophils in inflamed psoriatic skin (n = 30 fields from 10 patient samples). MFI of CXCR4 on peripheral neutrophils ( h ) and serum protein levels of CXCL12, IL-17A, and MPO ( i ) before and after treatment with anti-IL-17 inhibitor for 12 weeks. n = 5 biologically independent samples. Data are mean ± SD. Analyses: unpaired Student’s t-test in ( a) and ( c) ; The Spearman method in ( b ) and ( d ); Paired Student’s t-test in ( h ) and ( i ). The paired and unpaired Student’s t-test were conducted as two-sided tests. FMO, Fluorescence Minus One; HC, healthy control; MFI, mean fluorescence intensity; Pre, pre-treatment; Pso, psoriasis patients. Source data are provided as a Source Data file.

Article Snippet: CXCR4 hi neutrophils were also obtained by positive selection from total neutrophils using mouse CXCR4 MicroBeads (130-118-682, Miltenyi Biotec Inc.) according to the manufacturer’s protocol, as depicted below.

Techniques: Fluorescence, Western Blot, Expressing, Immunofluorescence, Staining, Control

Journal: Nature Communications

Article Title: CREB1-driven CXCR4 hi neutrophils promote skin inflammation in mouse models and human patients

doi: 10.1038/s41467-023-41484-3

Figure Lengend Snippet: a Representative images and quantification of different nucleus morphology of CXCR4 lo and CXCR4 hi neutrophils from healthy controls and psoriasis patients. Scale bar = 5 µm. b Flow cytometry analysis of key immune markers between peripheral CXCR4 lo and CXCR4 hi neutrophils from healthy controls and psoriasis patients (scale: log-transformed MFI). c Assessment of CXCR4 hi neutrophil survival by Annexin V and 7-AAD staining after 24 h of culture. d Measurement of ROS production by DHE fluorescence. e Quantification of NETs in CXCR4 lo and CXCR4 hi neutrophils from healthy controls and psoriasis patients. Phagocytosis of pHrodo Green E. coli by neutrophils was evaluated by immunofluorescence staining ( f ) and flow cytometry ( g ). Scale bar = 5 µm. h Degranulation of CXCR4 lo or CXCR4 hi neutrophils as assessed by CD63 expression. ELISA quantification ( i ) and representative immunoblots ( j ) for mature MMP-9 in the supernatant of CXCR4 lo and CXCR4 hi neutrophils from healthy controls and psoriasis patients that cultured for 24 h. GAPDH was analyzed from corresponding neutrophils. Blots for each antigen were processed in the same experiment in parallel. k Relative mRNA expression of pro-inflammatory mediators in CXCR4 lo and CXCR4 hi neutrophils from healthy controls and psoriasis patients. Data are mean ± SD (n = 6 biologically independent samples). The immunofluorescence staining was repeated three times independently with similar results. Two-way ANOVA with Tukey’s post hoc test was performed as two-sided analyses and adjusted for multiple comparisons in the statistical analyses. ns, not significant. HC, healthy control; MFI, mean fluorescence intensity; MMP-9, matrix metalloprotein 9; NETs; neutrophil extracellular traps; neu, neutrophils; Pso, psoriasis patients. Source data are provided as a Source Data file.

Article Snippet: CXCR4 hi neutrophils were also obtained by positive selection from total neutrophils using mouse CXCR4 MicroBeads (130-118-682, Miltenyi Biotec Inc.) according to the manufacturer’s protocol, as depicted below.

Techniques: Flow Cytometry, Transformation Assay, Staining, Fluorescence, Immunofluorescence, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Cell Culture, Control

Journal: Nature Communications

Article Title: CREB1-driven CXCR4 hi neutrophils promote skin inflammation in mouse models and human patients

doi: 10.1038/s41467-023-41484-3

Figure Lengend Snippet: a Volcano plot showing the number of differentially expressed genes (DEGs) between CXCR4 lo and CXCR4 hi neutrophils from healthy controls (n = 7) and psoriasis patients (n = 6). b Heatmap of DEGs between psoriatic CXCR4 lo and CXCR4 hi neutrophils related to neutrophil and immune function. c Enriched GO terms between CXCR4 lo and CXCR4 hi psoriatic neutrophils. d Gene set enrichment analysis showing enrichment of genes involved in glycolysis in psoriatic CXCR4 hi neutrophils. e Flow cytometry of glycolytic markers in CXCR4 lo and CXCR4 hi neutrophils from healthy controls and psoriasis patients. f Uptake of glucose (2-NBDG) in CXCR4 lo and CXCR4 hi neutrophils from healthy controls and psoriasis patients. g Extracellular lactate production in CXCR4 lo and CXCR4 hi neutrophils from healthy controls and psoriasis patients. Mean ± SD (n = 6 biologically independent samples/group). Two-way ANOVA with Tukey’s post hoc test was performed as two-sided analyses and adjusted for multiple comparisons in the statistical analyses. ns, not significant. HC, healthy control; MFI, mean fluorescence intensity; Pso, psoriasis patients; RNA-seq, RNA sequencing. Source data are provided as a Source Data file.

Article Snippet: CXCR4 hi neutrophils were also obtained by positive selection from total neutrophils using mouse CXCR4 MicroBeads (130-118-682, Miltenyi Biotec Inc.) according to the manufacturer’s protocol, as depicted below.

Techniques: Flow Cytometry, Control, Fluorescence, RNA Sequencing

Journal: Nature Communications

Article Title: CREB1-driven CXCR4 hi neutrophils promote skin inflammation in mouse models and human patients

doi: 10.1038/s41467-023-41484-3

Figure Lengend Snippet: a Representative images of adherent CXCR4 lo and CXCR4 hi neutrophils co-cultured with HMEC-1 cells. White arrows indicate HMEC-1 cells; red arrows indicate adherent neutrophils. Scale bar = 10 µm. b Relative mRNA expression of adhesion molecules in HMEC-1 cells with indicated treatment. Representative immunoblots ( c ) and qRT-PCR analysis ( d ) of tight junctions in HMEC-1 cells co-cultured with indicated treatment. e Stimulation of HMEC-1 cells with CXCR4 lo and CXCR4 hi neutrophils for 6 h, followed by analysis of leaked fluorescence intensity of FITC-dextran in a Transwell system. Representative immunoblots of HMEC-1 cells pre-incubated with LDHA inhibitor for 30 min following co-culture with psoriatic CXCR4 hi neutrophils ( f ) and qRT-PCR assessment of tight junction genes ( g ). h Analysis of released FITC-dextran in a Transwell system with HMEC-1 cells, pre-incubated with LDHA inhibitor for 30 min, followed by co-culture with psoriatic CXCR4 hi neutrophils. i Confocal images of CD31 (green) and GPR81 (purple) in psoriatic lesions (n = 6) with CD15 + CXCR4 hi neutrophils adjacent to GPR81 + vascular ECs. Scale bar = 50 µm, 20 µm. The result was repeated three times independently with similar results. HMEC-1 cells were transfected with GPR81 siRNA and subjected to indicated treatment, followed by Western blot ( j ), qRT-PCR ( k ), and FITC-dextran leakage ( l ). The immunoblotting samples shown are from the same experiment ( c , f and j ) and blots were processed in parallel. Mean ± SD (n = 6 biologically independent samples/group). Analyses: two-way ANOVA with Tukey’s post hoc test in ( b ), ( d ), ( e ), ( h ) and ( l ); One-way ANOVA with Tukey’s post hoc test in ( g ) and ( k ). One or two-way ANOVA tests were performed as two-sided analyses and adjusted for multiple comparisons in the statistical analyses. ns, not significant. HC, healthy control; HMEC-1 cells, human microvascular endothelial cells; Pso, psoriasis patients. Source data are provided as a Source Data file.

Article Snippet: CXCR4 hi neutrophils were also obtained by positive selection from total neutrophils using mouse CXCR4 MicroBeads (130-118-682, Miltenyi Biotec Inc.) according to the manufacturer’s protocol, as depicted below.

Techniques: Cell Culture, Expressing, Western Blot, Quantitative RT-PCR, Fluorescence, Incubation, Co-Culture Assay, Transfection, Control

Journal: Nature Communications

Article Title: CREB1-driven CXCR4 hi neutrophils promote skin inflammation in mouse models and human patients

doi: 10.1038/s41467-023-41484-3

Figure Lengend Snippet: a Expression of CXCR4 on neutrophils stimulated with IL-25, CXCL12, and TNF for 2 h was measured by flow cytometry. b Serum level of CXCL12 in healthy controls (n = 24) and psoriasis patients (n = 30) was detected by ELISA. c Expression of CXCR4 on neutrophils with indicated treatment was measured by flow cytometry. d Representative immunofluorescence staining of CD31 (yellow), Vimentin (green), and CXCL12 (purple) in inflamed psoriatic skin (n = 6). Scale bar = 50 µm, 20 µm. e Representative immunofluorescence staining of CD31 (yellow), CXCR4 (green), and CXCL12 (purple) in inflamed psoriatic skin (n = 6). Scale bar = 50 µm, 20 µm. QRT-PCR analysis ( f ) and flow cytometry analysis ( g ) of CXCR4 in healthy neutrophils with indicated treatments. h Representative immunofluorescence co-staining of CXCR4 (red) and LAMP1 (green) in neutrophils. Scale bar = 2 µm. i Examples of low colocalization values in a sample of CXCR4 hi and CXCR4 lo neutrophils of psoriasis patients stained for LCN2 (red) and CD63 (yellow) was visualized by ImageStream analysis. Images are from one representative experiment out of six. The scale bar indicates 7 μm. Isolated neutrophils were used for ( a ), ( c ), ( f ), and ( g ). Mean ± SD (n = 6 biologically independent samples/group). The immunofluorescence staining was repeated three times independently with similar results. Analyses: one-way ANOVA with Tukey’s post hoc test in ( a) and ( c) ; two-way ANOVA with Tukey’s post hoc test in ( f ) and ( g ); Unpaired Student’s t-test in ( b ). The unpaired Student’s t-test was conducted as two-sided tests. One or two-way ANOVA tests were performed as two-sided analyses and adjusted for multiple comparisons in the statistical analyses. ns, not significant. FMO, Fluorescence Minus One; HC, healthy control; LCN2, lipocalin 2; MFI, mean fluorescence intensity; Pso, psoriasis patients. Source data are provided as a Source Data file.

Article Snippet: CXCR4 hi neutrophils were also obtained by positive selection from total neutrophils using mouse CXCR4 MicroBeads (130-118-682, Miltenyi Biotec Inc.) according to the manufacturer’s protocol, as depicted below.

Techniques: Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Quantitative RT-PCR, Isolation, Fluorescence, Control

Journal: Nature Communications

Article Title: CREB1-driven CXCR4 hi neutrophils promote skin inflammation in mouse models and human patients

doi: 10.1038/s41467-023-41484-3

Figure Lengend Snippet: a Conceptual network diagram of genes showing the relationship between CREB1 and enrichment pathways, and heatmap of top transcription factors in CXCR4 hi neutrophils. The mRNA expression of CREB1 ( b ) and protein levels of p-CREB1 (s133) ( c ) in CXCR4 lo and CXCR4 hi neutrophils. d Flow cytometry analysis of p-CREB1 (s133) in neutrophils treated with indicated stimulation. e Co-localization of CXCR4 (green) and p-CREB1 (red) in neutrophils from healthy controls and psoriasis patients. Scale bar = 2 µm. Representative staining ( f ) and Western blot ( g ) of CBP (green) and p-CREB1 (red) in neutrophils treated with indicated stimulation for 2 h. h , i Healthy neutrophils were pre-treated with CREB1 inhibitor (KG-501, 300 μM) for 1 h, followed by indicated stimulation for 2 h. Flow cytometry analysis of the expression of membrane molecules ( h ), as well as glycolysis ( i ). j Representative co-staining of DNA (Hoechst), Cit-H3 (citrullinated histone-3, red), and MPO (myeloperoxidase, green) to assess NETs formation in psoriatic neutrophils in vitro after pretreatment with KG-501 (300 μM) for 1 h. Scale bar = 20 µm. k Western blot of dHL-60 cells transfected with CREB1 siRNA and subjected to the indicated treatment. l Depiction of the CREB1 binding site in the CXCR4 promoter for ChIP assay. m The luciferase activities of wild type CXCR4 and CXCR4 with the CREB1-binding site mutant were determined by luciferase reporter gene assays in dHL-60 cells. Isolated neutrophils were used for ( b ), ( d ), ( h ), and ( i ), and whole blood was used for ( c ). Mean ± SD (n = 6 biologically independent samples). The immunofluorescence staining was repeated three times independently with similar results. Blots for each antigen were processed in the same experiment in parallel. Two-way ANOVA with Tukey’s post hoc test ( b, c, h, i , and m ) and one-way ANOVA with Tukey’s post hoc test ( d ) were performed as two-sided analyses and adjusted for multiple comparisons in the statistical analyses. ns, not significant. HC, healthy control; MFI, mean fluorescence intensity; NETs, neutrophil extracellular traps; Pso, psoriasis patients. Source data are provided as a Source Data file.

Article Snippet: CXCR4 hi neutrophils were also obtained by positive selection from total neutrophils using mouse CXCR4 MicroBeads (130-118-682, Miltenyi Biotec Inc.) according to the manufacturer’s protocol, as depicted below.

Techniques: Expressing, Flow Cytometry, Staining, Western Blot, Membrane, In Vitro, Transfection, Binding Assay, Luciferase, Mutagenesis, Isolation, Immunofluorescence, Control, Fluorescence

Journal: Nature Communications

Article Title: CREB1-driven CXCR4 hi neutrophils promote skin inflammation in mouse models and human patients

doi: 10.1038/s41467-023-41484-3

Figure Lengend Snippet: a Relative mRNA expressions of CXCR4 and CXL12 in mice tissues was evaluated by qRT-PCR. n = 6 mice. b Proportion of Ly6G + CXCR4 hi neutrophils in mice skin was determined by flow cytometry. n = 6 mice. c Serum level of CXCL12 in control and IMQ-treated mice was detected by ELISA. n = 6 mice. d Schematic diagram of mouse experimental protocol. IMQ mice were injected intraperitoneally with an anti-Ly6G antibody every other day and then injected subcutaneously with fresh isolated homologous Ly6G + CXCR4 lo or Ly6G + CXCR4 hi neutrophils daily. e Phenotype and representative H&E staining of IMQ-treated mice in indicated groups on day 5. Images are representative of six individual mouse per group. Control group was topically applied with Vaseline cream. Bar = 200 µm. n = 6 mice. f Epidermal thickness was assessed by H&E. n = 20 vision fields from 6 mice. g Representative immunofluorescence staining of CD31 (red) in inflamed skin. Scale bar = 50 µm. n = 6 mice. h Quantification of the dermal vascular area in the H&E-stained sections. n = 10 vision fields from 6 mice. i Relative mRNA expressions of inflammatory cytokines in mice tissues. n = 6 mice. The immunofluorescence staining was repeated three times independently with similar results. Mean ± SD. Analyses: unpaired Student’s t-test in ( a ), ( b ), and ( c ); One-way ANOVA with Tukey’s post hoc test in ( f ), ( h ), and ( i ). The unpaired Student’s t-test was conducted as two-sided tests. One-way ANOVA test was performed as two-sided analyses and adjusted for multiple comparisons in the statistical analyses. ns, not significant; IMQ, imiquimod. Source data are provided as a Source Data file.

Article Snippet: CXCR4 hi neutrophils were also obtained by positive selection from total neutrophils using mouse CXCR4 MicroBeads (130-118-682, Miltenyi Biotec Inc.) according to the manufacturer’s protocol, as depicted below.

Techniques: Quantitative RT-PCR, Flow Cytometry, Control, Enzyme-linked Immunosorbent Assay, Injection, Isolation, Staining, Cream, Immunofluorescence

Journal: Nature Communications

Article Title: CREB1-driven CXCR4 hi neutrophils promote skin inflammation in mouse models and human patients

doi: 10.1038/s41467-023-41484-3

Figure Lengend Snippet: a Phenotype and H&E staining of IMQ-treated mice in different treatment groups. Images are representative of six individual mice per group. The mice in the control group was topically applied with vaseline cream. Bar = 200 µm. n = 6 mice. b Epidermal thickness as assessed by H&E staining. n = 20 vision fields from 6 mice. c Proportion of Ly6G + CXCR4 hi neutrophils in inflamed skin was determined by flow cytometry. n = 6 mice. d Representative immunofluorescence staining of CD31 (red) in lesional skin of indicated groups. Scale bar = 50 µm. n = 6 mice. e Quantification of the dermal vascular area in the H&E-stained sections. n = 10 vision fields from 6 mice. f Quantification of extracted Evans blue dye in inflamed skin in indicated groups. n = 6 mice. g Relative mRNA expressions of inflammatory cytokines in mice tissues. n = 6 mice. The immunofluorescence staining was repeated three times independently with similar results. Mean ± SD. One-way ANOVA with Tukey’s post hoc test was performed as two-sided analyses and adjusted for multiple comparisons in the statistical analyses. ns, not significant; IMQ, imiquimod. Source data are provided as a Source Data file.

Article Snippet: CXCR4 hi neutrophils were also obtained by positive selection from total neutrophils using mouse CXCR4 MicroBeads (130-118-682, Miltenyi Biotec Inc.) according to the manufacturer’s protocol, as depicted below.

Techniques: Staining, Control, Cream, Flow Cytometry, Immunofluorescence

Journal: Nature Communications

Article Title: CREB1-driven CXCR4 hi neutrophils promote skin inflammation in mouse models and human patients

doi: 10.1038/s41467-023-41484-3

Figure Lengend Snippet: This study provides insights into the development and role of CXCR4 hi neutrophils in skin inflammation. Compared to CXCR4 lo neutrophils, CXCR4 hi neutrophils exhibit increased capacity for NETs formation, phagocytosis, and degranulation, as well as higher expression of pro-inflammatory mediators, supported by activated glycolysis. CXCR4 expression on neutrophils is induced by CXCL12, IL-25, and TNF, which requires de novo mRNA and protein synthesis and intracellular protein transport, but is not stored in neutrophil granules and regulated by degranulation. CREB1 is activated by phosphorylation on Ser-133 upon stimulation, enabling interaction with its coactivator protein CBP to initiate transcription of CREB-responsive genes, thereby contributing to CXCR4 expression and CXCR4 hi neutrophils in skin inflammation. Our data further emphasize the crucial role of CXCR4 hi neutrophils in promoting vascular remodeling through lactate and MMP-9 release (B), facilitating immune cell infiltration into tissues, and increasing inflammatory responses (A). Moreover, we identify the CXCR4/CXCL12 axis as a potential therapeutic target for inflammatory skin diseases, with a focus on CREB1, CXCR4/CXCL12, or the formation of NETs. CBP, CREB-binding protein; EC, endothelial cell; MMP9, matrix metallopeptidase 9; NETs; neutrophil extracellular traps; PAD4 i, PAD4 inhibitor. Created with BioRender.com.

Article Snippet: CXCR4 hi neutrophils were also obtained by positive selection from total neutrophils using mouse CXCR4 MicroBeads (130-118-682, Miltenyi Biotec Inc.) according to the manufacturer’s protocol, as depicted below.

Techniques: Expressing, Phospho-proteomics, Binding Assay

Journal: Molecular medicine reports

Article Title: miR‑137 decreases proliferation, migration and invasion in rheumatoid arthritis fibroblast‑like synoviocytes.

doi: 10.3892/mmr.2017.8225

Figure Lengend Snippet: Figure 4. CXCL12 is a direct target of miR‑137 in RA‑FLS. (A) Predicted binding site for miR‑137 in the 3'UTR of Wt‑CXCL12; mutations in the binding sites in the Mut‑CXCL12 sequence are also indicated. (B) Relative luciferase activity of in RA‑FLS co‑transfected with either Wt‑CXCL12 or Mut‑CXCL12 3'UTR reporter plasmid and either miR‑137 mimic or miR‑NC. **P<0.01 vs. miR‑NC. The expression levels of CXCL12 (C) mRNA and (D) protein were detected in FLS isolated the RA model rat group and the normal control group by RT‑PCR and western blotting, respectively. GAPDH was used as an internal control. **P<0.01 vs. normal FLS. (E and F) RA‑FLS were transfected with either miR‑137 mimic or miR‑NC, and CXCL12 (E) mRNA and (F) protein expression levels were determined. GAPDH was used as an internal control. **P<0.01 vs. miR‑NC. CXCL12, C‑X‑C motif chemokine ligand 12; FLS, fibroblast‑like synoviocytes; miR, microRNA; Mut, mutant; NC, negative control; RA, rheumatoid arthritis; UTR, untranslated region; Wt, wild‑type.

Article Snippet: Membranes were blocked with 5% non‐fat milk in Tris‐buffered saline (TBS; Sigma‐Aldrich; Merck KGaA) for 1 h at room temperature, and incubated overnight at 4 ̊C with the following primary antibodies: Mouse monoclonal anti-human CXCL12 (1:1,000; cat no. sc‐74271) and mouse monoclonal anti‐human GAPDH (1:5,000; cat no. sc-293335); both were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Binding Assay, Sequencing, Luciferase, Activity Assay, Plasmid Preparation, Expressing, Isolation, Control, Western Blot, Transfection, Mutagenesis, Negative Control

Journal: BMC Cancer

Article Title: COUP-TFI modifies CXCL12 and CXCR4 expression by activating EGF signaling and stimulates breast cancer cell migration

doi: 10.1186/1471-2407-14-407

Figure Lengend Snippet: Sequences of primers used in this study

Article Snippet: A goat polyclonal antibody against human CXCL12 (R&D Systems AF-310-NA), rabbit polyclonal antibody against CXCR4 (Abcam Inc. ab2074), mouse monoclonal antibody against human CXCR7/RDC1 (R&D Systems clone 11G8; MAB42273), a rabbit polyclonal antibody against COUP-TFI (Abcam Inc. ab11954) and a rabbit polyclonal antibody against HA epitope (Santa Cruz sc-805) were used for the immunofluorescence and western blot assays.

Techniques:

Journal: BMC Cancer

Article Title: COUP-TFI modifies CXCL12 and CXCR4 expression by activating EGF signaling and stimulates breast cancer cell migration

doi: 10.1186/1471-2407-14-407

Figure Lengend Snippet: COUP-TFI modifies the expression of the CXCL12 signaling axis in MCF-7 cells. (A) Characterization of the control and COUP clones. An immunofluorescence cytochemistry assay was used to detect the relative expression of HA/COUP-TFI or COUP-TFI proteins in the control (Cont.) and COUP clones. The cells were fixed and processed for immunofluorescence as described in Methods; the nuclei were stained with DAPI. (B) CXCL12 , CXCR4 , and CXCR7 mRNAs were quantified by a real-time PCR analysis from two independent MCF-7 control and COUP clones. The results were normalized to GAPDH mRNA used as an internal control. The results were expressed as the relative mRNA expression level of CXCL12 , CXCR4 , or CXCR7 . Data are the mean values ± SEM of at least three independent experiments. The asterisks indicate significant differences ( p < 0.05) between the control and COUP clones. (C) The amount of intracellular HA/COUP-TFI, COUP-TFI, CXCL12, CXCR4, and CXCR7 protein was determined from whole-cell extracts of the different MCF-7 clones and compared to total ERK. A representative western blot is shown. (D) The control and COUP clones were fixed, and an immunofluorescence cytochemistry assay was used to detect the relative expression of CXCL12, CXCR4, and CXCR7 proteins. Staining with DAPI is also presented to visualize the nucleus of the cells.

Article Snippet: A goat polyclonal antibody against human CXCL12 (R&D Systems AF-310-NA), rabbit polyclonal antibody against CXCR4 (Abcam Inc. ab2074), mouse monoclonal antibody against human CXCR7/RDC1 (R&D Systems clone 11G8; MAB42273), a rabbit polyclonal antibody against COUP-TFI (Abcam Inc. ab11954) and a rabbit polyclonal antibody against HA epitope (Santa Cruz sc-805) were used for the immunofluorescence and western blot assays.

Techniques: Expressing, Control, Clone Assay, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Western Blot

Journal: BMC Cancer

Article Title: COUP-TFI modifies CXCL12 and CXCR4 expression by activating EGF signaling and stimulates breast cancer cell migration

doi: 10.1186/1471-2407-14-407

Figure Lengend Snippet: COUP-TFI modulates the chromatin structure of the CXCL12 and CXCR4 gene promoters. The FAIRE assay was performed as described in Methods. Real-time PCR was performed to monitor the enrichment of DNA corresponding to the proximal promoter of the CXCL12 (A) , the CXCR4 (B) and the CXCR7 (C) genes relative to the input chromatin from the control (Cont.) and COUP clones. The data are from triplicate samples and are representative of three separate experiments. The asterisk indicates significant differences ( p < 0.05) between the control and COUP clones.

Article Snippet: A goat polyclonal antibody against human CXCL12 (R&D Systems AF-310-NA), rabbit polyclonal antibody against CXCR4 (Abcam Inc. ab2074), mouse monoclonal antibody against human CXCR7/RDC1 (R&D Systems clone 11G8; MAB42273), a rabbit polyclonal antibody against COUP-TFI (Abcam Inc. ab11954) and a rabbit polyclonal antibody against HA epitope (Santa Cruz sc-805) were used for the immunofluorescence and western blot assays.

Techniques: Real-time Polymerase Chain Reaction, Control, Clone Assay

Journal: BMC Cancer

Article Title: COUP-TFI modifies CXCL12 and CXCR4 expression by activating EGF signaling and stimulates breast cancer cell migration

doi: 10.1186/1471-2407-14-407

Figure Lengend Snippet: Estrogenic regulation of CXCL12 and CXCR4 in control and COUP clones. Control (Cont.) and COUP clones were treated with ethanol (EtOH) as the vehicle or E2 10 −8 M and ICI 10 −6 M alone or both together for 48 h. The CXCL12 (A) and CXCR4 (B) relative mRNA levels were monitored by a real-time PCR analysis, normalized to GAPDH mRNA as the internal control, and were expressed as the relative mRNA expression of CXCL12 or CXCR4 . Data are the mean ± SEM of at least three independent experiments. The asterisks indicate significant differences ( p < 0.05) between the untreated and treated control clones. The pound sign indicates significant differences ( p < 0.05) between the untreated and treated COUP clones.

Article Snippet: A goat polyclonal antibody against human CXCL12 (R&D Systems AF-310-NA), rabbit polyclonal antibody against CXCR4 (Abcam Inc. ab2074), mouse monoclonal antibody against human CXCR7/RDC1 (R&D Systems clone 11G8; MAB42273), a rabbit polyclonal antibody against COUP-TFI (Abcam Inc. ab11954) and a rabbit polyclonal antibody against HA epitope (Santa Cruz sc-805) were used for the immunofluorescence and western blot assays.

Techniques: Control, Clone Assay, Real-time Polymerase Chain Reaction, Expressing

Journal: BMC Cancer

Article Title: COUP-TFI modifies CXCL12 and CXCR4 expression by activating EGF signaling and stimulates breast cancer cell migration

doi: 10.1186/1471-2407-14-407

Figure Lengend Snippet: The effect of COUP-TFI on the CXCL12/CXCR4 axis is mediated by EGF/EGFR activation. (A) The relative expression of EGF and EGFR mRNA was monitored by a real-time PCR analysis using MCF-7 control (Cont.) and COUP clones. The results were normalized against GAPDH as the internal control and are expressed as the mean EGF or EGFR mRNA/GAPDH mRNA ratio ± SEM of at least three independent experiments. The asterisks indicate significant differences ( p < 0.05) between the control and COUP clones. (B) ERK activation was examined in the MCF-7 control (Cont.) and COUP clones after a 5- or 10-min stimulation with EGF (10 −9 M) or CXCL12 (200 ng/mL). Western blots were performed using antibodies against phospho-ERK (P-ERK) and total ERK (ERK1/2); a representative western blot is presented. The importance of EGFR-specific signaling and general ERK signaling on CXCL12 (C) and CXCR4 (D) regulation was assayed by treating the cells with EGF (10 −9 M), AG1478 (25 μM), or U0126 (25 μM) for 48 h. The CXCL12 and CXCR4 relative mRNA levels were monitored by the real-time PCR analysis, normalized to GAPDH mRNA as the internal control, and were expressed as the relative mRNA expression of CXCL12 or CXCR4 . Data are the mean ± SEM of at least three independent experiments. The asterisks indicate significant differences ( p < 0.05) between the untreated and treated control clones. The pound sign indicates significant differences ( p < 0.05) between the untreated and treated COUP clones.

Article Snippet: A goat polyclonal antibody against human CXCL12 (R&D Systems AF-310-NA), rabbit polyclonal antibody against CXCR4 (Abcam Inc. ab2074), mouse monoclonal antibody against human CXCR7/RDC1 (R&D Systems clone 11G8; MAB42273), a rabbit polyclonal antibody against COUP-TFI (Abcam Inc. ab11954) and a rabbit polyclonal antibody against HA epitope (Santa Cruz sc-805) were used for the immunofluorescence and western blot assays.

Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Control, Clone Assay, Western Blot

Journal: BMC Cancer

Article Title: COUP-TFI modifies CXCL12 and CXCR4 expression by activating EGF signaling and stimulates breast cancer cell migration

doi: 10.1186/1471-2407-14-407

Figure Lengend Snippet: COUP-TFI overexpression influences cellular responses to CXCL12. (A) The relative growth of the control (Cont.) and COUP clones was assayed with or without CXCL12 treatment for 7 days. The basal and CXCL12-induced cell growth were evaluated by MTT assays (n = 6) and determined in three independent experiments. The results are expressed as the relative cell number obtained when the control cells were treated with the vehicle control. Significant differences between the unstimulated control cells and the other conditions ( p < 0.05) are indicated with an asterisk. Significant differences between stimulated control cells and stimulated COUP clones ( p < 0.05 ) are indicated with a pound sign. (B) The migratory capacity of control (Cont.) and COUP clones was analyzed. The cells were maintained in phenol red-free DMEM/2.5% dsFBS for 48 h and then seeded in phenol red-free DMEM/0.5% dsFBS in the upper chamber of a PET 8-μm pore insert. The cells were allowed to migrate for 24 h toward the phenol red-free DMEM/2.5% dsFBS medium complemented or not with CXCL12 (200 ng/mL) and AMD3100 (50 μM). (C) CXCL12 was also added to the culture medium in the upper chamber prior to migration. The results are expressed as the mean ± SEM of the relative number of migratory cells compared to the basal migration of the control cells measured in three independent experiments. The asterisks indicate significant differences ( p < 0.05) from the basal migration of the control clones. The pound sign indicates significant differences ( p < 0.05) between two conditions linked by black lines.

Article Snippet: A goat polyclonal antibody against human CXCL12 (R&D Systems AF-310-NA), rabbit polyclonal antibody against CXCR4 (Abcam Inc. ab2074), mouse monoclonal antibody against human CXCR7/RDC1 (R&D Systems clone 11G8; MAB42273), a rabbit polyclonal antibody against COUP-TFI (Abcam Inc. ab11954) and a rabbit polyclonal antibody against HA epitope (Santa Cruz sc-805) were used for the immunofluorescence and western blot assays.

Techniques: Over Expression, Control, Clone Assay, Migration

Journal: BMC Cancer

Article Title: COUP-TFI modifies CXCL12 and CXCR4 expression by activating EGF signaling and stimulates breast cancer cell migration

doi: 10.1186/1471-2407-14-407

Figure Lengend Snippet: Box plots of CXCR4, CXCR7, CXCL12, and COUP-TFI mRNA expression in breast cancer and normal tissue. CXCR4 (A) , CXCR7 (B) , CXCL12 (C) , and COUP-TFI (D) mRNA expression was measured by real-time PCR in 23 normal breast tissue samples (NT), in 20 SBR grades 1 and 2, and in 19 SBR grades 3. The expression level was normalized by 18S RNA expression and analyzed with IQ5 software (Bio-Rad). The data are presented as whisker plots in which the horizontal bar represents the median, the grey boxes are the 25 th and 75 th percentiles, the vertical bar is the standard deviation, and the plus signs are the extreme points. All the Mann-Whitney tests were performed with Minitab 16 software, and the p value is indicated on the different graphs (ns denotes non-significant).

Article Snippet: A goat polyclonal antibody against human CXCL12 (R&D Systems AF-310-NA), rabbit polyclonal antibody against CXCR4 (Abcam Inc. ab2074), mouse monoclonal antibody against human CXCR7/RDC1 (R&D Systems clone 11G8; MAB42273), a rabbit polyclonal antibody against COUP-TFI (Abcam Inc. ab11954) and a rabbit polyclonal antibody against HA epitope (Santa Cruz sc-805) were used for the immunofluorescence and western blot assays.

Techniques: Expressing, Real-time Polymerase Chain Reaction, RNA Expression, Software, Whisker Assay, Standard Deviation, MANN-WHITNEY

Journal: bioRxiv

Article Title: Rotary jet-spun porous microfibers as scaffolds for stem cells delivery to central nervous system injury

doi: 10.1101/239194

Figure Lengend Snippet: MSC compatibility with PRM scaffolds. (A) Characterization of PRM scaffolds by scanning electron microscopy. (B) BrdU and phalloidin immunostaining of MSCs on coverslips or PRM scaffolds. Cells were able to adhere and incorporate BrdU into DNA (proliferating cells) in either condition (n = 2, experiment in triplicates). Scale bar = 100 μm. (C) After seven days in culture, MTT assay showed similar survival of MSCs when cells were cultured on coverslips or PRM scaffolds (p > 0.05, Student’s t test, n = 5). (D) TUNEL assay showed that MSCs cultured on coverslips or PRM scaffolds did not undergo apoptosis (n = 2, experiment in triplicates). Scale bar = 100 μm. (E) MSCs cultured on coverslips or PRM scaffolds were induced to differentiate in osteocytes and adipocytes. After 3 weeks, cells were stained with Oil Red (staining of lipid vesicles in red) or Alizarin Red (staining of calcium deposits in red). MSCs cultured on PRM scaffolds preserved multipotency (n = 2, experiment in triplicates). Upper scale bar = 50 μm; lower scale bar = 200 μm. (F) PRM induced a 50% increase in CXCL12 secretion by MSCs ( p < 0.05, Student’s t test, n = 5). MSCs: mesenchymal stem cells, PRM: polymeric rough microfibers, BrdU: 5-bromo-2’-deoxyuridine, MTT: 3-(4,5-dimethylthiazol-yl)2,5-diphenyltetrazolium bromide, TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, DAPI: 4’,6-diamidino-2-phenylindole.

Article Snippet: CXCL 12 in conditioned media was quantified using Mouse CXCL12 DuoSet ELISA (R&D systems, Minneapolis, USA).

Techniques: Electron Microscopy, Immunostaining, MTT Assay, Cell Culture, TUNEL Assay, Staining, End Labeling

Journal: The Biochemical journal

Article Title: Stromal cell-derived factor-1 (CXCL12) activates integrins by direct binding to an allosteric ligand-binding site (site 2) of integrins without CXCR4.

doi: 10.1042/BCJ20170867

Figure Lengend Snippet: (a) Docking model of SDF1-αvβ3 (inactive) interaction. The position of site 2 peptide of β3 is shown in blue. The simulation predicted amino acid residues involved in integrin binding (Table 1), including Lys24, Lys27, and Lys43 of SDF1. (b) Peptide derived from site 2 (Fc-β3) binds to SDF1. SDF1 binding to immobilized Fc-β3 peptide, scrambled peptide (Fc-β3scr), or Fc was measured by using anti-SDF1 antibody. Data are shown as means +/− SEM of triplicate experiments. (c) SDF1 activates soluble αvβ3 in cell-free conditions. The binding of soluble αvβ3 to immobilized γC399tr was measured as described in the methods section. Data are shown as means +/− SEM of triplicate experiments. (d) Time course of SDF1-induced activation of soluble αvβ3. Data are shown as means +/− SEM of triplicate experiments.

Article Snippet: After unbound SDF1 was removed by rinsing the wells with PBS, bound SDF1 was measured using anti-SDF1 antibody (R&D systems MAB350, mouse monoclonal antibody to Lys22-Lys89 of SDF1) and HRP-conjugated anti-mouse IgG.

Techniques: Binding Assay, Derivative Assay, Activation Assay

Journal: The Biochemical journal

Article Title: Stromal cell-derived factor-1 (CXCL12) activates integrins by direct binding to an allosteric ligand-binding site (site 2) of integrins without CXCR4.

doi: 10.1042/BCJ20170867

Figure Lengend Snippet: Amino acid residues involved in the interaction between SDF1 and integrin αvβ3 (site 2).

Article Snippet: After unbound SDF1 was removed by rinsing the wells with PBS, bound SDF1 was measured using anti-SDF1 antibody (R&D systems MAB350, mouse monoclonal antibody to Lys22-Lys89 of SDF1) and HRP-conjugated anti-mouse IgG.

Techniques:

Journal: The Biochemical journal

Article Title: Stromal cell-derived factor-1 (CXCL12) activates integrins by direct binding to an allosteric ligand-binding site (site 2) of integrins without CXCR4.

doi: 10.1042/BCJ20170867

Figure Lengend Snippet: The binding of FITC-labeled γC399tr to the cell-surface αvβ3 on β3-CHO cells in the presence of SDF and Fc-β3 peptide was measured using flow cytometry as described in the methods section. MFI= median fluorescent intensity. Data are shown as means +/− SEM of triplicate experiments. (a) SDF1 activates integrin αvβ3 on β3-CHO cells in a dose-dependent manner (n=4). (b) Fc-β3 peptide, not Fc-β3scr or Fc, suppresses SDF1-mediated activation of integrin αvβ3. (c) AMD3100, a CXCR4 inhibitor, does not affect SDF1-mediated αvβ3 activation in an allosteric manner. (d) SDF1 mutations in the predicted site 2-binding interface of SDF1 are defective in αvβ3 activation. (e) SDF1 fully activates cell-surface αvβ3 in 1 min and the activation lasts for 1 h.

Article Snippet: After unbound SDF1 was removed by rinsing the wells with PBS, bound SDF1 was measured using anti-SDF1 antibody (R&D systems MAB350, mouse monoclonal antibody to Lys22-Lys89 of SDF1) and HRP-conjugated anti-mouse IgG.

Techniques: Binding Assay, Labeling, Flow Cytometry, Activation Assay

Journal: The Biochemical journal

Article Title: Stromal cell-derived factor-1 (CXCL12) activates integrins by direct binding to an allosteric ligand-binding site (site 2) of integrins without CXCR4.

doi: 10.1042/BCJ20170867

Figure Lengend Snippet: Parent CHO cells (α5β1+) were incubated with FITC-labeled ECM ligands (the fibronectin fragment FN8–11 specific to α5β1) in the presence of SDF1 (20 μg/ml) and/or Fc-β3 peptide (200 μg/ml) and the binding of the ligands was measured in flow cytometry. MFI= median fluorescent intensity. Data are shown as means +/− SEM of triplicate experiments. (a) SDF1 activates integrin α5β1 on CHO cells in a dose-dependent manner (n=4). (b) Fc-β3 peptide, not Fc-β3scr or Fc, suppresses SDF1-mediated activation of integrin α5β1. (c) AMD3100, a CXCR4 inhibitor, does not affect SDF1-mediated integrin α5β1 activation in an allosteric manner. (d) SDF1 mutations in the predicted site 2-binding interface of SDF1 are defective in α5β1 activation. (e) SDF1 fully activates cell-surface α5β1 in 1 min and the activation lasts for 1 h.

Article Snippet: After unbound SDF1 was removed by rinsing the wells with PBS, bound SDF1 was measured using anti-SDF1 antibody (R&D systems MAB350, mouse monoclonal antibody to Lys22-Lys89 of SDF1) and HRP-conjugated anti-mouse IgG.

Techniques: Incubation, Labeling, Binding Assay, Flow Cytometry, Activation Assay

Journal: The Biochemical journal

Article Title: Stromal cell-derived factor-1 (CXCL12) activates integrins by direct binding to an allosteric ligand-binding site (site 2) of integrins without CXCR4.

doi: 10.1042/BCJ20170867

Figure Lengend Snippet: α4-CHO cells were incubated with FITC-labeled ECM ligands (the fibronectin fragment H120 specific to α4β1) in the presence of SDF1 (20 μg/ml) and/or Fc-β3 peptide (200 μg/ml) and the binding of the ligands was measured in flow cytometry. MFI= median fluorescent intensity. Data are shown as means +/− SEM of triplicate experiments. (a) SDF1 activates integrin α4β1 on α4-CHO cells in a dose-dependent manner (n=4). (b) Fc-β3 peptide, not Fc-β3scr or Fc, suppresses SDF1-mediated activation of integrin α4β1. (c) AMD3100, a CXCR4 inhibitor, does not affect SDF1-mediated integrin α4β1 activation in an allosteric manner. (d) SDF1 mutations in the predicted site 2-binding interface of SDF1 are defective in α4β1 activation. (e) SDF1 fully activates cell-surface α4β1 in 1 min and the activation lasts for 1 h.

Article Snippet: After unbound SDF1 was removed by rinsing the wells with PBS, bound SDF1 was measured using anti-SDF1 antibody (R&D systems MAB350, mouse monoclonal antibody to Lys22-Lys89 of SDF1) and HRP-conjugated anti-mouse IgG.

Techniques: Incubation, Labeling, Binding Assay, Flow Cytometry, Activation Assay