Journal: Nature Cell Biology

Article Title: Peritumoural adipose tissue drives immune evasion in colorectal cancer via adipose–mesenchymal transformation

doi: 10.1038/s41556-026-01885-0

Figure Lengend Snippet: a . Representative images of MC38-OVA (left) and tumour growth (right) in C57BL/6J mice ( n = 5). b . Representative images of CT26 tumours (left) and tumour growth (right) in BALB/c mice ( n = 5). c . Schematic diagram illustrating the working principle of ATS-GNP adipocytolysis. ATS-GNP, containing a CaCO 3 core, is receptor-mediated for endocytosis. Upon reaching the acidic environment, ATS-GNP releases CO 2 gas, disrupting the adipose cell membrane. d . Tumour growth of MC38 tumours in C57BL/6J, BALB/c-Nude (left), and NSG (right) mice ( n = 5). e . Representative images of E0771 tumours at day 16 of the experiment in C57BL/6J mice following removal of PAT ( n = 5). f . Tumour weights of E0771 tumours at day 16 in C57BL/6J mice with the removal of PAT ( n = 5). g . Representative flow cytometry plots of CD45 + cells gated on live cells in MC38-OVA tumours. h . Violin plots showing the expression of CXCR7 across all cell types in CRC patients, analysed using the Kruskal-Wallis test. i . Editing strategy for constructing Cxcl12 fl/fl cKO mouse. j . Experimental design for constructing Control and Cxcl12 fl/fl cKO mice bearing MC38-OVA tumours near PAT. k . Western blot analysis for verifying the knockout efficiency of Cxcl12 in PAT of Control mice and Cxcl12 fl/fl cKO mice. l . RT-qPCR analysis for verifying the Cxcl12 knockout efficiency in liver, spleen, uterus, and tumour tissues from Control mice and Cxcl12 fl/fl cKO mice ( n = 6). Data represent ≥ 3 independent experiments. P -values were calculated using two-way ANOVA with Tukey’s correction for multiple comparisons ( a -right, b -right, and d ), two-way ANOVA with Bonferroni’s correction for multiple comparisons ( f ), or a two-sided, unpaired Student’s t -test ( l ). Graphs display mean ± SD ( a , b , d , f , l ). Panels created with BioRender : c , i and j , Huaiqiang, J. https://biorender.com/cy7rgjm (2026).

Article Snippet: For drug treatment, a CXCL12 neutralizing antibody (Merck Millipore) and an anti-mouse PD-1 antibody (Bio X Cell) were administered via intraperitoneal injection.

Techniques: Membrane, Flow Cytometry, Expressing, Control, Western Blot, Knock-Out, Quantitative RT-PCR

Journal: Nature Cell Biology

Article Title: Peritumoural adipose tissue drives immune evasion in colorectal cancer via adipose–mesenchymal transformation

doi: 10.1038/s41556-026-01885-0

Figure Lengend Snippet: a , Cell–cell communication analysis based on ligand–receptor interactions (top six) between stromal cells and lymphocytes in tVAT (left) and a comparison between tVAT, dVAT and tumour (right). b , Marked CXCL12–CXCR4 interactions among CD8 + T cells, CD4 + T cells, B cells, plasma cells and stromal cell populations in tVAT, dVAT and tumour. The width of the lines represents the probability of communication. c , Violin plots showing the expression of CXCL12 (top) and CXCR4 (bottom) across all cell types in patients with CRC. d , Violin plots comparing the expression of CXCL12 in dVAT versus tVAT (top) and tumour versus tVAT (bottom) in patients with CRC, analysed using a two-sided Wilcoxon test. e , Experimental design for the PAT C57BL/6J mouse model treated with IgG or anti-CXCL12 antibody (left), and representative MC38 tumour images at day 16 of the experiment (right) ( n = 5). f , Tumour growth (left) and tumour weights (right) of MC38 tumours at day 16 of the experiment in C57BL/6J mice ( n = 5). g , Representative MC38 tumour images (left) and tumour growth (right) of experiments in control and Cxcl12 fl/fl cKO mice ( n = 6). h , Tumour weights of MC38 tumours in control and Cxcl12 fl/fl cKO mice at day 16 of the experiment ( n = 6). i , Flow cytometry analysis of the infiltration of various CXCR4 + immune cells in MC38 tumours in Control and Cxcl12 fl/fl cKO mice ( n = 6). j , Schematic diagram of the chemotaxis assay using T cells as ‘sensors’ and conditional medium as a ‘sink’ (left), and the aggregated trajectories of control or CXCL12-induced T cells migrating for 1 h (right). k , Quantitative analysis of CXCR4 + CD45.1 + T cells in MC38 tumours with and without removal of PAT or contralateral inguinal adipose tissue (control) by flow cytometry ( n = 5). Data represent ≥3 independent experiments. Statistical significance was assessed by a two-sided permutation test ( a ), two-sided unpaired Student’s t -test ( f right, h and i ), one-way analysis of variance (ANOVA) with Tukey’s correction for multiple comparisons ( k right) or two-way ANOVA ( f left and g right). Graphs display mean ± s.d. ( f – i , k ). Panels created with BioRender : e and k , Huaiqiang, J. https://biorender.com/ovq2e39 (2026).

Article Snippet: For drug treatment, a CXCL12 neutralizing antibody (Merck Millipore) and an anti-mouse PD-1 antibody (Bio X Cell) were administered via intraperitoneal injection.

Techniques: Comparison, Clinical Proteomics, Expressing, Control, Flow Cytometry, Chemotaxis Assay

Journal: Nature Cell Biology

Article Title: Peritumoural adipose tissue drives immune evasion in colorectal cancer via adipose–mesenchymal transformation

doi: 10.1038/s41556-026-01885-0

Figure Lengend Snippet: a . UMAP plot of VAT-associated stromal cells in tVAT and dVAT from CRC patients. The 8 clusters, labelled by inferred cell types, are denoted by colour. b . Dot plot showing RNA expression of marker genes used to define pAC, adCAF, and APC subclusters. Circle size represents the log-normalized P -value, while colour intensity indicates the log-transformed mean expression of marker genes. c . Sorting strategy for isolating adCAFs from mouse PAT by flow cytometry. d . RNA sequencing analysis of adCAF markers in sorted adCAF-enriched stromal cells and non-adCAF stromal cells ( n = 4). Statistical significance was assessed using a two-sided, unpaired Student’s t -test. e . RNA sequencing analysis of adipogenesis markers in sorted adCAF-enriched stromal cells and non-adCAF stromal cells ( n = 4). Statistical significance was assessed using a two-sided, unpaired Student’s t -test. f . Representative images of the morphology of sorted adCAF-enriched stromal cells and non-adCAF stromal cells. Scale bar = 500 μm. g . Cross-tissue interactions analysis based on ligand-receptor pairs between adCAFs and tumour cells (left) and a comparison between tVAT and dVAT (right). A two-sided permutation test was used to determine the significance of pathways. h . Western blot analysis of CXCL12 protein expression in sorted adCAF-enriched stromal cells and non-adCAF stromal cells ( n = 3). The data are presented as a box-and-whisker graph (bounds of box: first to third quartile, bottom and top line: minimum to maximum, central line: median) for ( d - e ).

Article Snippet: For drug treatment, a CXCL12 neutralizing antibody (Merck Millipore) and an anti-mouse PD-1 antibody (Bio X Cell) were administered via intraperitoneal injection.

Techniques: RNA Expression, Marker, Transformation Assay, Expressing, Flow Cytometry, RNA Sequencing, Comparison, Western Blot, Whisker Assay

Journal: Nature Cell Biology

Article Title: Peritumoural adipose tissue drives immune evasion in colorectal cancer via adipose–mesenchymal transformation

doi: 10.1038/s41556-026-01885-0

Figure Lengend Snippet: a , UMAP of all stromal cells in tVAT, dVAT, tumour and normal from patients with CRC, with ten clusters labelled by inferred cell types. Major lineages included ASCs, pACs, CAFs, pericytes (PCs) and mesothelial cells (Mesos). b , Heatmap displaying the distribution of eight stromal cell subtypes across different tissue types. c , UMAP of eight subsets of VAT-associated stromal cells in tVAT and dVAT from patients with CRC, including ASCs, pACs and adCAFs. d , Beeswarm plot showing the distribution and abundance of VAT-associated stromal cell types in Nhoods between tVAT and dVAT. e , Stack plot displaying the abundance of the eight VAT-associated stromal cell subsets in dVAT and tVAT. f , Heatmap showing the RNA expression of various marker genes in VAT-associated stromal cell types, including ASC/pAC markers, CAF markers, cytokines and stromal markers. g , Representative multiplex immunofluorescence images showing the presence of adCAFs in tVAT samples from patients with CRC. Scale bar, 10 μm. DAPI, 4,6-diamidino-2-phenylindole. h , Relative expression levels of multiple cytokines and protumoural factors in adCAF-enriched stromal cells ( n = 4) and non-adCAF stromal cells ( n = 4) derived from the PAT of mice xenograft models. The data are presented as a box-and-whisker graph (bounds of box show first to third quartile, bottom and top line show minimum to maximum and the central line shows the median). i , Cell–cell communication analysis based on ligand–receptor pairs (top six) between adCAFs and lymphocytes in tVAT (left) and a comparison between tVAT and dVAT (right). j , RT–qPCR (left) and ELISA (right) detecting the RNA expression and protein secretion of CXCL12 in sorted adCAF-enriched stromal cells and non-adCAF stromal cells from PAT of mice. Data represent ≥3 independent experiments. All data are shown as mean ± s.d. and statistical significance was assessed by a two-sided, unpaired Wilcoxon test ( h ), two-sided permutation test ( i ) and Student’s t -test ( j ).

Article Snippet: For drug treatment, a CXCL12 neutralizing antibody (Merck Millipore) and an anti-mouse PD-1 antibody (Bio X Cell) were administered via intraperitoneal injection.

Techniques: RNA Expression, Marker, Multiplex Assay, Immunofluorescence, Expressing, Derivative Assay, Whisker Assay, Comparison, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Nature Cell Biology

Article Title: Peritumoural adipose tissue drives immune evasion in colorectal cancer via adipose–mesenchymal transformation

doi: 10.1038/s41556-026-01885-0

Figure Lengend Snippet: a . UMAP of all cells in tVAT and dVAT from 5 CRC patients, with 16 clusters labelled by inferred cell types. Major lineages included various immunocytes, VAT-associated stromal cells, adipocytes (ACs), endothelial cells (ECs), pericytes (PCs) and mesothelial cells (Mesos). b . Heatmap of representative marker genes across all cell populations. c . UMAP of 6 subsets of ACs in tVAT and dVAT from 5 CRC patients. d . Beeswarm plot showing the distribution and abundance of ACs in Nhoods between tVAT and dVAT. e . Box plot to compare the abundance of ACs in dVAT ( n = 5) and tVAT ( n = 5). P -values were calculated using a two-side Wilcoxon test. f . Cell-cell communication analysis based on ligand-receptor interactions (top 3) between CD8 + T cells and adipocytes, and between CD8 + T cells and VAT-associated stromal cells in tVAT. A two-sided permutation test was used to determine the significance of pathways. Only significant ligand-receptor pairs ( P < 0.05) are shown. Dot size encodes the interaction score, and colour scale indicates the communication probability. g . Violin plots showing the expression of CXCL12, VAT-associated markers, and CAF-associated markers in adipocytes and VAT-associated stromal cells from CRC patients. h . UMAP of 8 subsets of VAT-associated stromal cells in tVAT and dVAT from 5 CRC patients, including APCs, pACs and adipocyte-derived cancer-associated fibroasts (adCAFs). The 8 clusters, labelled by inferred cell types, are denoted by colour. i . Beeswarm plot showing the distribution and abundance of VAT-associated stromal cell types in Nhoods between tVAT ( n = 5) and dVAT ( n = 5). j . Box plot to compare the abundance of VAT-associated stromal cells in dVAT and tVAT. P -values were calculated using a two-side Wilcoxon test. k . Heatmap showing the RNA expression patterns of representative marker genes across all cell populations, including CAF markers, stromal markers, APC/pAC markers, white/brown/beige fat markers, and cytokines and growth markers. The data are presented as a box-and-whisker graph (bounds of box: first to third quartile, bottom and top line: minimum to maximum, central line: median) for ( e , j ).

Article Snippet: For drug treatment, a CXCL12 neutralizing antibody (Merck Millipore) and an anti-mouse PD-1 antibody (Bio X Cell) were administered via intraperitoneal injection.

Techniques: Marker, Expressing, Derivative Assay, RNA Expression, Whisker Assay

Journal: Nature Cell Biology

Article Title: Peritumoural adipose tissue drives immune evasion in colorectal cancer via adipose–mesenchymal transformation

doi: 10.1038/s41556-026-01885-0

Figure Lengend Snippet: a , Experimental design for constructing Control and Mdk DTR cKO mice bearing MC38 tumours near PAT, followed by αPD-1 therapy. b , c , Representative MC38 tumour images ( b ), tumour weights ( c left) and tumour growth ( c right) in control and Mdk DTR cKO mice treated with IgG or anti-PD-1 ( n = 6). d , Flow cytometry analysis of the infiltration of immunocytes, including T cells, CD4 + T cells, CD8 + T cells, and tumour-specific CD8 + T cells, CXCR4 + immunocytes, CXCR4 + T cell, CXCR4 + CD4 + T cell, CXCR4 + CD8 + T cell and CXCR4 + tumour-specific T cell in MC38-OVA tumours from the four treatment groups ( n = 6). e , f , Representative MC38 tumour images ( e ) and tumour weights ( f ) of the experiment in mice treated with anti-CXCL12 and/or anti-PD-1 ( n = 5). g , Representative MRI image of CRC tumour and corresponding tVAT area region of CR and non-CR patients pre- and post-immuno-chemoradiotherapy. The yellow area represents the tVAT area, whereas the red area denotes the tumour region. Note that the mass visible in the intestinal lumen (top right) is faecal material. h , Pre-treatment tVAT area difference based on 3D Slicer between CR ( n = 30) and non-CR ( n = 37) patients. The data are presented as a box-and-whisker graph (bounds of box show first to third quartile, bottom and top line show minimum to maximum and the central line shows the median). i , ROC plot of response predicting ability of pre-treatment PAT area in immuno-chemoradiotherapy of proficient mismatch repair patients with CRC, compared with conventional indexes, including CPS, TPS, CEA and CA199 ( n = 67) with optimal cutoff. j , Comparison of pCR ratio in tVAT high and low group according to the optimal cutoff. k , Graphical abstract depicting how tumours reshape the stromal environment in tVAT and how tVAT competes for immunocytes from the tumour to promote immune escape. Data represent ≥3 independent experiments. Statistical significance was assessed using a two-sided, unpaired Student’s t -test ( d , h ), one-way ANOVA with Tukey’s correction for multiple comparisons ( c left, f ) or two-way ANOVA with Tukey’s correction for multiple comparisons ( c right). Graphs display mean ± s.d. ( c , d , f , h ). Panels created with BioRender : a and k , Huaiqiang, J. https://biorender.com/e5jwcye (2026).

Article Snippet: For drug treatment, a CXCL12 neutralizing antibody (Merck Millipore) and an anti-mouse PD-1 antibody (Bio X Cell) were administered via intraperitoneal injection.

Techniques: Control, Flow Cytometry, Whisker Assay, Comparison

Journal: Nature Cell Biology

Article Title: Peritumoural adipose tissue drives immune evasion in colorectal cancer via adipose–mesenchymal transformation

doi: 10.1038/s41556-026-01885-0

Figure Lengend Snippet: a . Editing strategy for constructing Mdk DTR mouse. b . Flow cytometry representative plots and bar graphs for verifying the elimination efficiency of adCAFs (FAP + PDGFRB + MDK + stromal cells) in PAT of Control mice and Mdk DTR cKO mice. c . Experimental design for combination therapy with αCXCL12 and αPD-1 in mice bearing MC38-OVA tumours near PAT. d . MC38 tumour weights of the experiment in mice treated with αCXCL12 and/or αPD-1 ( n = 6). e . Flow cytometry analysis of the infiltration of immunocytes, including T cells, CD4 + T cells, CD8 + T cells, and tumour-specific CD8 + T cells in MC38-OVA tumours from the 4 treatment groups ( n = 6). f . Flow cytometry analysis of the infiltration of CXCR4 + immunocytes, including CXCR4 + T cell, CXCR4 + CD4 + T cell and CXCR4 + CD8 + T cell in MC38-OVA tumours from the 4 treatment groups ( n = 6). g . Boxplot of difference of tVAT area in CR and non-CR patients with T3 or T4 stage separately. The data are presented as a box-and-whisker graph (bounds of box: first to third quartile, bottom and top line: minimum to maximum, central line: median). h . ROC plot of prediction ability of tVAT area in patients with T3 or T4 stage separately. Data represent ≥ 3 independent experiments. P -values were calculated using a two-sided, unpaired Student’s t -test ( b , e - g ) and two-way ANOVA with Tukey’s correction for multiple comparisons ( d ). Graphs display mean ± SD ( b , d , e - g ). Panels created with BioRender : a and c , Huaiqiang, J. https://biorender.com/33r2gmm (2026).

Article Snippet: For drug treatment, a CXCL12 neutralizing antibody (Merck Millipore) and an anti-mouse PD-1 antibody (Bio X Cell) were administered via intraperitoneal injection.

Techniques: Flow Cytometry, Control, Whisker Assay

Journal: Nature Cell Biology

Article Title: Peritumoural adipose tissue drives immune evasion in colorectal cancer via adipose–mesenchymal transformation

doi: 10.1038/s41556-026-01885-0

Figure Lengend Snippet: a . Experimental design for combination therapy with AMD3100 and αPD-1 in mice bearing MC38-OVA tumours near subcutaneous PAT. b . Representative MC38 tumour images of the mice treated with AMD3100 and/or αPD-1 ( n = 5). c . Representative MC38 tumour weights of the mice treated with AMD3100 and/or αPD-1 ( n = 5). d . Representative MC38 tumour growth of the mice treated with AMD3100 and/or αPD-1 ( n = 5). e . Representative MC38 tumour images in MC38-OVA caecal orthotopic tumour-bearing mice treated with AMD3100 and/or αPD-1 ( n = 5). f . Representative MC38 tumour weights in MC38-OVA caecal orthotopic tumour-bearing mice treated with AMD3100 and/or αPD-1 ( n = 5). g . Representative MC38 Bioluminescence Images in MC38-OVA caecal orthotopic tumour-bearing mice treated with AMD3100 and/or αPD-1 ( n = 5). h . Representative MC38 tumour images in MC38-OVA caecal orthotopic tumour-bearing mice treated with αCXCL12 and/or αPD-1 ( n = 5). i . Representative MC38 tumour weights in MC38-OVA caecal orthotopic tumour-bearing mice treated with αCXCL12 and/or αPD-1 ( n = 5). j . Representative MC38 Bioluminescence Images in MC38-OVA caecal orthotopic tumour-bearing mice treated with αCXCL12 and/or αPD-1 ( n = 5). k . Flow cytometry analysis of the infiltration of immunocytes, including T cells, CD4 + T cells, CD8 + T cells, and tumour-specific CD8 + T cells in MC38-OVA tumours from 4 treatment groups ( n = 5). l . Flow cytometry analysis of the infiltration of CXCR4 + immunocytes, including CXCR4 + T cell, CXCR4 + CD4 + T cell and CXCR4 + CD8 + T cell in MC38-OVA tumours from 4 treatment groups ( n = 5). Data represent ≥ 3 independent experiments. P -values were calculated using a two-sided, unpaired Student’s t -test ( c , f , i , k , l ) and two-way ANOVA with Tukey’s correction for multiple comparisons ( d ). Graphs display mean ± SD ( c - d , f , i , k - l ). Panel created with BioRender : a , Huaiqiang, J. https://biorender.com/wbcolts (2026).

Article Snippet: For drug treatment, a CXCL12 neutralizing antibody (Merck Millipore) and an anti-mouse PD-1 antibody (Bio X Cell) were administered via intraperitoneal injection.

Techniques: Flow Cytometry

Journal: Journal of Translational Medicine

Article Title: CXCR4/CXCL12 axis promotes lymphatic metastasis in tongue squamous cell carcinoma via PI3K/AKT signaling pathway

doi: 10.1186/s12967-025-06707-9

Figure Lengend Snippet: CXCR4 and CXCL12 expression in TSCC tissues and their association with patient survival. a Representative immunohistochemical staining of CXCR4 in normal tongue epithelium, lymphoid tissue, and TSCC tissue (left panels) and quantification of IHC scores (right panel). Scale bars, 100 μm. b Representative immunohistochemical staining of CXCL12 in normal tongue epithelium, lymphoid tissue, and TSCC tissue (left panels) and quantification of IHC scores (right panel). Scale bars, 100 μm. c Kaplan–Meier survival analysis of TSCC patients stratified by CXCR4 expression. d Kaplan–Meier survival analysis of TSCC patients stratified by CXCL12 expression. Data are presented as mean ± SD. ****p < 0.0001 (compared to Control)

Article Snippet: Where indicated, CM was pre-incubated with a CXCL12 neutralizing antibody (R&D Systems, Cat# MAB350, 10 μg/ml) or an isotype control IgG antibody (R&D Systems, Cat# MAB002) for 1 h at 37 °C before use.

Techniques: Expressing, Immunohistochemical staining, Staining, Control

Journal: Journal of Translational Medicine

Article Title: CXCR4/CXCL12 axis promotes lymphatic metastasis in tongue squamous cell carcinoma via PI3K/AKT signaling pathway

doi: 10.1186/s12967-025-06707-9

Figure Lengend Snippet: CXCR4 and CXCL12 serve as predictive biomarkers for lymphatic metastasis in TSCC patients. a Forest plot showing the results of multivariate Cox regression analysis for overall survival in TSCC patients. b Receiver operating characteristic (ROC) curve analysis of CXCR4 expression for predicting lymph node metastasis in TSCC patients. c ROC curve analysis of CXCL12 expression for predicting lymph node metastasis in TSCC patients. d Nomogram for predicting the probability of lymph node metastasis in TSCC patients incorporating CXCR4 expression, CXCL12 expression, tumor size, tumor depth, differentiation, and age. e Calibration curve of the nomogram for predicting lymph node metastasis in TSCC patients. f Kaplan–Meier survival analysis of TSCC patients stratified by risk score based on the nomogram

Article Snippet: Where indicated, CM was pre-incubated with a CXCL12 neutralizing antibody (R&D Systems, Cat# MAB350, 10 μg/ml) or an isotype control IgG antibody (R&D Systems, Cat# MAB002) for 1 h at 37 °C before use.

Techniques: Expressing

Journal: Journal of Translational Medicine

Article Title: CXCR4/CXCL12 axis promotes lymphatic metastasis in tongue squamous cell carcinoma via PI3K/AKT signaling pathway

doi: 10.1186/s12967-025-06707-9

Figure Lengend Snippet: CXCR4 regulates TSCC cell proliferation, migration, invasion, and EMT. a Cell proliferation assessed by CCK-8 assay in CAL-27 cells with different CXCR4 expression levels (Control, shControl, shRNA-CXCR4-KD, and shRNA-CXCR4-OE). b Colony formation assay of CAL-27 cells with different CXCR4 expression levels. Representative images (left) and quantification (right). c Transwell migration assay of CAL-27 cells with different CXCR4 expression levels. Representative images (left) and quantification (right). Scale bars, 100 μm. d Transwell invasion assay of CAL-27 cells with different CXCR4 expression levels. Representative images (left) and quantification (right). Scale bars, 100 μm. e Quantification of apoptotic cells measured by flow cytometry. f Representative flow cytometry plots showing apoptosis in CAL-27 cells with different CXCR4 expression levels. g Western blot analysis of EMT markers (E-cadherin, N-cadherin, and Vimentin) in CAL-27 cells with different CXCR4 expression levels. h qRT-PCR analysis of EMT markers in CAL-27 cells with different CXCR4 expression levels. i Wound healing assay showing the effect of CXCL12 on CAL-27 cell migration. Representative images (left) and quantification (right). Scale bars, 100 μm. j Real-time cell analysis (RTCA) showing the effect of CXCL12 on CAL-27 cell migration. Data are presented as mean ± SD from three independent experiments. *p < 0.05 , **p < 0.01 , ***p < 0.001 , ****p < 0.0001 (compared to Control or Serum-Free); # p < 0.05 , ## p < 0.01 , #### p < 0.0001 (compared to shControl or Blank-Serum); && p < 0.01 , &&&& p < 0.0001 (compared to shControl- CXCR4-KD)

Article Snippet: Where indicated, CM was pre-incubated with a CXCL12 neutralizing antibody (R&D Systems, Cat# MAB350, 10 μg/ml) or an isotype control IgG antibody (R&D Systems, Cat# MAB002) for 1 h at 37 °C before use.

Techniques: Migration, CCK-8 Assay, Expressing, Control, shRNA, Colony Assay, Transwell Migration Assay, Transwell Invasion Assay, Flow Cytometry, Western Blot, Quantitative RT-PCR, Wound Healing Assay, Cell Analysis

Journal: Journal of Translational Medicine

Article Title: CXCR4/CXCL12 axis promotes lymphatic metastasis in tongue squamous cell carcinoma via PI3K/AKT signaling pathway

doi: 10.1186/s12967-025-06707-9

Figure Lengend Snippet: The PI3K/AKT pathway mediates CXCR4/CXCL12-induced TSCC progression. a Western blot analysis of PI3K, p-AKT, and AKT in CAL-27 cells treated with CXCL12 (100 ng/ml) for the indicated times (0, 5, 15, 30, and 60 min). Representative blots (left) and quantification (right). b Western blot analysis of PI3K, p-AKT, and AKT in CAL-27 cells treated with different concentrations of CXCL12 (0, 10, 50, 100, and 200 ng/ml) for 30 min. Representative blots (left) and quantification (right). c Western blot analysis of PI3K, p-AKT, and AKT in CAL-27 cells treated with CXCL12 (100 ng/ml), AMD3100 (10 μM), or CXCL12 + AMD3100. Representative blots (left) and quantification (right). d Immunofluorescence staining of STAT3 in CAL-27 cells treated with CXCL12 (100 ng/ml) or vehicle control. Scale bars, 20 μm. e Western blot analysis of p-AKT and AKT in CAL-27 cells treated with CXCL12 (100 ng/ml), LY294002 (20 μM), or CXCL12 + LY294002. Representative blots (left) and quantification (right). f Transwell migration assay of CAL-27 cells treated with CXCL12 (100 ng/ml), AMD3100 (10 μM), or CXCL12 + AMD3100. Representative images (left) and quantification (right). Scale bars, 100 μm. g Transwell invasion assay of CAL-27 cells treated with CXCL12 (100 ng/ml), LY294002 (20 μM), or CXCL12 + LY294002. Representative images (left) and quantification (right). Scale bars, 100 μm. h Western blot analysis of EMT markers (E-cadherin, N-cadherin, and Vimentin) in CAL-27 cells treated with CXCL12 (100 ng/ml), AMD3100 (10 μM), LY294002 (20 μM), or combinations. Representative blots (left) and quantification (right). Data are presented as mean ± SD from three independent experiments. *p < 0.05 , **p < 0.01 , ***p < 0.001 , ****p < 0.0001 (compared to 0 or Control); # p < 0.05 , ### p < 0.001 , #### p < 0.0001 (compared to AMD3100 or CXCL12); && p < 0.01 , &&& p < 0.001 , &&&& p < 0.0001 (compared to CXCL12 or CXCL12 + AMD3100)

Article Snippet: Where indicated, CM was pre-incubated with a CXCL12 neutralizing antibody (R&D Systems, Cat# MAB350, 10 μg/ml) or an isotype control IgG antibody (R&D Systems, Cat# MAB002) for 1 h at 37 °C before use.

Techniques: Western Blot, Immunofluorescence, Staining, Control, Transwell Migration Assay, Transwell Invasion Assay

Journal: Journal of Translational Medicine

Article Title: CXCR4/CXCL12 axis promotes lymphatic metastasis in tongue squamous cell carcinoma via PI3K/AKT signaling pathway

doi: 10.1186/s12967-025-06707-9

Figure Lengend Snippet: CXCR4/CXCL12 signaling promotes lymphangiogenesis in TSCC. a Cell proliferation of human lymphatic endothelial cells (HLECs) treated with CXCL12 (100 ng/ml) or vehicle control, assessed by CCK-8 assay. b Wound healing assay showing the effect of CXCL12 on HLEC migration. Representative images (left) and quantification (right). Scale bars, 100 μm. c Tube formation assay of HLECs treated with CXCL12 (100 ng/ml) or vehicle control. Representative images (left) and quantification (right). Scale bars, 100 μm. d Western blot analysis of lymphangiogenic factors (VEGF-C, VEGFR-3, and Prox1) in HLECs treated with CXCL12 (100 ng/ml) or vehicle control. Representative blots (left) and quantification (right). e Western blot analysis of PI3K, p-AKT, and AKT in HLECs treated with CXCL12 (100 ng/ml) or vehicle control. Representative blots (left) and quantification (right). f Tube formation assay of HLECs treated with CXCL12 (100 ng/ml), LY294002 (20 μM), or CXCL12 + LY294002. Representative images (left) and quantification (right). Scale bars, 100 μm. g Schematic representation of the co-culture system used to investigate the interaction between TSCC cells and HLECs. h Transwell migration assay of HLECs co-cultured with CAL-27 cells with different CXCR4 expression levels. Representative images (left) and quantification (right). Scale bars, 100 μm. Data are presented as mean ± SD from three independent experiments. ***p < 0.001 , ****p < 0.0001 (compared to Control); #### p < 0.0001 (compared to CXCL12)

Article Snippet: Where indicated, CM was pre-incubated with a CXCL12 neutralizing antibody (R&D Systems, Cat# MAB350, 10 μg/ml) or an isotype control IgG antibody (R&D Systems, Cat# MAB002) for 1 h at 37 °C before use.

Techniques: Control, CCK-8 Assay, Wound Healing Assay, Migration, Tube Formation Assay, Western Blot, Co-Culture Assay, Transwell Migration Assay, Cell Culture, Expressing

Journal: Journal of Translational Medicine

Article Title: CXCR4/CXCL12 axis promotes lymphatic metastasis in tongue squamous cell carcinoma via PI3K/AKT signaling pathway

doi: 10.1186/s12967-025-06707-9

Figure Lengend Snippet: CXCR4/CXCL12 axis promotes TSCC growth and lymphatic metastasis in vivo. a Schematic representation of the orthotopic TSCC mouse model. b Tumor growth curves of CAL-27 cells with different CXCR4 expression levels (Control, CXCR4-KD, CXCR4-OE) or treated with AMD3100. c Tumor weight at the endpoint. d Lymph node metastasis rates in different groups. e Representative images of H&E staining of cervical lymph nodes at low magnification (100 ×). Scale bars, 200 μm. f Representative images of H&E staining of cervical lymph nodes at high magnification (400 ×). Scale bars, 50 μm. g Immunohistochemical staining of CXCR4 and CXCL12 in primary tumors from different groups. Scale bars, 100 μm. h Immunohistochemical staining of EMT markers (E-cadherin, N-cadherin, and Vimentin) in primary tumors from different groups. Scale bars, 100 μm. i Quantification of lymphatic vessel density (LVD) in primary tumors from different groups. Data are presented as mean ± SD. ****p < 0.0001 (compared to Control); ### p < 0.001 , #### p < 0.0001 (compared to CXCR4-KD); &&&& p < 0.0001 (compared to CXCR4-OE)

Article Snippet: Where indicated, CM was pre-incubated with a CXCL12 neutralizing antibody (R&D Systems, Cat# MAB350, 10 μg/ml) or an isotype control IgG antibody (R&D Systems, Cat# MAB002) for 1 h at 37 °C before use.

Techniques: In Vivo, Expressing, Control, Staining, Immunohistochemical staining

Journal: Journal of Translational Medicine

Article Title: CXCR4/CXCL12 axis promotes lymphatic metastasis in tongue squamous cell carcinoma via PI3K/AKT signaling pathway

doi: 10.1186/s12967-025-06707-9

Figure Lengend Snippet: Transcriptomic analysis of CXCR4/CXCL12-regulated gene expression in TSCC. a Principal component analysis (PCA) of RNA-seq data from CAL-27 cells with different CXCR4 expression levels (Control, CXCR4-KD, and CXCR4-OE). b Volcano plot showing differentially expressed genes (DEGs) between Control and CXCR4-KD CAL-27 cells. c Volcano plot showing DEGs between Control and CXCR4-OE CAL-27 cells. d Heatmap of DEGs between Control and CXCR4-KD CAL-27 cells. e Heatmap of DEGs between Control and CXCR4-OE CAL-27 cells. f qRT-PCR validation of selected DEGs ( PI3K3CA , AKT1 , PTEN , CXCL12 , and CXCR4 ) in CAL-27 cells with different CXCR4 expression levels. g Western blot validation of selected DEGs in CAL-27 cells with different CXCR4 expression levels. Representative blots (left) and quantification (right). h Proposed mechanistic model illustrating how the CXCR4/CXCL12 axis promotes TSCC lymphatic metastasis through the PI3K/AKT pathway. Data are presented as mean ± SD from three independent experiments. * p < 0.05 , ** p < 0.01 , *** p < 0.001 , ****p < 0.0001 (compared to Control); ## p < 0.01 , ### p < 0.001 , #### p < 0.0001 (compared to CXCR4-OE)

Article Snippet: Where indicated, CM was pre-incubated with a CXCL12 neutralizing antibody (R&D Systems, Cat# MAB350, 10 μg/ml) or an isotype control IgG antibody (R&D Systems, Cat# MAB002) for 1 h at 37 °C before use.

Techniques: Gene Expression, RNA Sequencing, Expressing, Control, Quantitative RT-PCR, Biomarker Discovery, Western Blot

Journal: Journal of Translational Medicine

Article Title: CXCR4/CXCL12 axis promotes lymphatic metastasis in tongue squamous cell carcinoma via PI3K/AKT signaling pathway

doi: 10.1186/s12967-025-06707-9

Figure Lengend Snippet: Autocrine and Paracrine Effects of TSCC-Derived CXCL12 and Generalizability of CXCR4/CXCL12 Signaling. a Transwell migration assay of CAL-27 cells stimulated with their own conditioned medium (CM). b Transwell invasion assay of CAL-27 cells under the same conditions as ( a ). c Tube formation assay of Human Lymphatic Endothelial Cells (HLECs) stimulated with CAL-27 conditioned medium. d Transwell migration assay of HSC-3 TSCC cells. e Transwell invasion assay of HSC-3 cells under the same conditions as ( d ). f Western blot analysis of phosphorylated AKT (P-AKT) and total AKT in HSC-3 cells. g ELISA quantification of CXCL12 levels in serum-free medium (Control) and CAL-27 conditioned medium (CAL-27 CM) after 48 h of culture. Representative images are shown for A-E. Bar graphs represent quantification of migrated/invaded cells or relative length of tubes, presented as mean ± SD from three independent experiments. *** p < 0.001, **** p < 0.0001 (compared to control); ### p < 0.001, #### p < 0.0001 (compared to CM or CXCL12 group); &&& p < 0.001, &&&& p < 0.0001 (compared to CM + IgG group); ns = not significant

Article Snippet: Where indicated, CM was pre-incubated with a CXCL12 neutralizing antibody (R&D Systems, Cat# MAB350, 10 μg/ml) or an isotype control IgG antibody (R&D Systems, Cat# MAB002) for 1 h at 37 °C before use.

Techniques: Derivative Assay, Transwell Migration Assay, Transwell Invasion Assay, Tube Formation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Control

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

Journal: Developmental cell

Article Title: Meningeal macrophages inhibit chemokine signaling in pre-tumor cells to suppress mouse medulloblastoma initiation.

doi: 10.1016/j.devcel.2023.08.033

Figure Lengend Snippet: Figure 6. CXCL4 inhibits tumorigenesis by antagonizing CXCL12/CXCR4-stimulated GNP chemotaxis and proliferation (A) Chemotactic responses of purified GNPs. Images show crystal violet stained (red) migrated GNPs on the bottom of a transwell insert. (B) Quantification of the number of migrated cells per high power field (HPF) for each condition (n = 3 samples per condition, 2 technical replicates each) (*p < 0.05 compared with Shh #p < 0.05 compared with Shh+CXCL12, one-way ANOVA). (C) GNP proliferation assay cultures stained for EdU (red), pCXCR4 (green), Ki67 (white), and 40,6-diamidino-2-phenylindole (DAPI) (blue).

Article Snippet: To assess the effect of the CXCL12 ligand on tumourigenesis, CXCL12 neutralizing antibodies (MAB310) and KLH control antibodies (MAB002) (R&D) were reconstituted at 0.5 mg/mL in sterile 1X PBS at injected at a dosage of 2.5 mg/kg.

Techniques: Chemotaxis Assay, Staining, Proliferation Assay

Journal: Developmental cell

Article Title: Meningeal macrophages inhibit chemokine signaling in pre-tumor cells to suppress mouse medulloblastoma initiation.

doi: 10.1016/j.devcel.2023.08.033

Figure Lengend Snippet: Figure 7. Norrin regulated meninges-GNP crosstalk in tumor initiation (A) In the developing cerebellum, GNP proliferation and retention in the pre- tumor niche is regulated by the coordinated activity of meningeal-derived chemokines: pro-tumorigenic CXCL12 and anti-tumorigenic CXCL4. CXCL12 activates CXCR4 in GNPs (red) to promote proliferation, and CXCL4 functions to prevent the CXCL12/CXCR4 interaction. Norrin signaling in meningeal endothelial cells (mECs) maintains meningeal macrophage (mMF) homeo- stasis, characterized by Lyve-1hi Mrc1hi CXCL4hi expression. (B) In the absence of Norrin, mMFs downregulate CXCL4, leading to a de- repression of CXCL12/CXCR4 signaling in GNPs, which, in turn, promotes cell- cycle progression and retention in the pre-tumor niche, which collectively re- sults in increased preneoplastic lesion formation and tumor incidence.

Article Snippet: To assess the effect of the CXCL12 ligand on tumourigenesis, CXCL12 neutralizing antibodies (MAB310) and KLH control antibodies (MAB002) (R&D) were reconstituted at 0.5 mg/mL in sterile 1X PBS at injected at a dosage of 2.5 mg/kg.

Techniques: Activity Assay, Derivative Assay, Expressing

Journal: International Journal of Biological Sciences

Article Title: Specific overexpression of SIRT1 in mesenchymal stem cells rescues hematopoiesis niche in BMI1 knockout mice through promoting CXCL12 expression

doi: 10.7150/ijbs.63876

Figure Lengend Snippet: Defects of HSPC niche composed by osteoblastic cells in Bmi1 KO mice. (A) 3 rd passage of bone-derived mesenchymal progenitor cells (BdMPC) from 4-week-old WT and KO mice treated in osteoblast induction medium containing β-glycerophosphate and ascorbic acid for 4 days before ALP staining. ALP + osteoblasts shown in red after ALP staining. (B) The percentage of ALP + cell area versus total cell growth area was counted. n=3 samples. (C) Alp gene transcription levels in bulk mRNA from 3 rd passage of BdMPCs treated with osteoblast induction medium for 4 days in (A). n=3 samples. (D) Gene transcription levels of niche factors, including macrophage colony-stimulating factor ( M-csf ), N-cadherin, osteopontin ( Opn ), Cxcl12 , Angiopoietin 1 ( Ang1 ), stem cell factor ( Scf ), Jagged 1 ( Jag1 ), Vcam1 , integrin β1 and β2 , in 3 rd passage of BdMPCs from 3-week-old WT and KO mice were tested by qPCR. n=3 samples. (E-F) Protein levels of cellular (E) and secreted (F) CXCL12 and Ang1 were measured in cellular protein lysates and condensed medium supernatant from 3rd passage of BdMPCs by WB, respectively. (G) WT and KO BM cells were cultured in conditioned medium for 48 h before flow analysis. Conditioned medium (CM): Mixture of 50% (v/v) fresh culture medium with 50% conditioned medium collected from WT or KO BdMPC culture. The bar graph in right panel shows frequencies of LSK cells in cultured BM cells. n=4 samples. (H) Representative H&E-stained tibial paraffin sections from WT and KO mice with intratibial injection of vehicle or CXCL12. n=4 mice. (I) Flow analysis of LSK cells in BM of mice in Fig. H. *p<0.05, **p<0.01, ***p<0.001. Data are mean ± SD; Unpaired two-tailed Student t test in (B), (C) and (D). One-way ANOVA with Tukey's post-hoc test in (G) and (I).

Article Snippet: 5×10 6 total bone marrow cells were suspended in 3 ml conditioned culture medium (1.5 ml collected supernatant (serum free) mixed with 1.5 ml fresh culture medium containing 30% FBS) with 20 μg/ml control IgG or 20 μg/ml anti-CXCL12 neutralizing antibody (Millipore; #MABC184) or 20 μg/ml angiopoietin blocking peptide (MyBioSource; #MBS8243845) and seeded in each well of Nunc TM non-treated 6-well plate from Thermo Scientific.

Techniques: Derivative Assay, Staining, Cell Culture, Injection, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: Specific overexpression of SIRT1 in mesenchymal stem cells rescues hematopoiesis niche in BMI1 knockout mice through promoting CXCL12 expression

doi: 10.7150/ijbs.63876

Figure Lengend Snippet: Sirt1 overexpression in MPCs partially prevented bone loss and CXCL12 reduction in Bmi1 KO mice. (A) BdMPCs from 1-m-old WT mice were transfected with Bmi1-pcDNA3.1 or vector plasmid using electroporation; 48 h post transfection, Bmi1 and Sirt1 expression was detected using immunofluorescence (IF) staining. (B) IF staining for Bmi1 and CXCL12 protein expression by BdMPCs as in (A). (C) Coronal sections of µCT 3D reconstruction of tibial metaphyseal region and (D) analyses of trabecular bone volume (BV/TV) and number (Tb.N) of tibiae from litters of 6-week-old WT, Sirt1 transgenic (TG), Bmi1 knockout (KO) and double mutant (KO-TG) mice. n=6 mice/group. (E) IHC staining for Collagen type-I (Col-I) expression on paraffin sections of proximal tibiae and (F) analyses of Col-1 + area in total tissue area (Col-I + A/T.A). n=6 mice/group. (G) Cxcl12 gene transcription levels in 3 rd passage of BdMPCs from 6-week-old WT, TG, KO and KO-TG mice. n=3 mice/group. *p<0.05, **p<0.01, ***p<0.001. Data are mean ± SD. One-way ANOVA with Tukey's post-hoc test.

Article Snippet: 5×10 6 total bone marrow cells were suspended in 3 ml conditioned culture medium (1.5 ml collected supernatant (serum free) mixed with 1.5 ml fresh culture medium containing 30% FBS) with 20 μg/ml control IgG or 20 μg/ml anti-CXCL12 neutralizing antibody (Millipore; #MABC184) or 20 μg/ml angiopoietin blocking peptide (MyBioSource; #MBS8243845) and seeded in each well of Nunc TM non-treated 6-well plate from Thermo Scientific.

Techniques: Over Expression, Transfection, Plasmid Preparation, Electroporation, Expressing, Immunofluorescence, Staining, Transgenic Assay, Knock-Out, Mutagenesis, Immunohistochemistry

Journal: International Journal of Biological Sciences

Article Title: Specific overexpression of SIRT1 in mesenchymal stem cells rescues hematopoiesis niche in BMI1 knockout mice through promoting CXCL12 expression

doi: 10.7150/ijbs.63876

Figure Lengend Snippet: Dietary supplementation of resveratrol partially rescues osteogenic defects in Bmi1 KO mice. (A) Coronal sections of µCT 3D reconstruction of tibial metaphyseal region and (B) analyses of trabecular bone volume (BV/TV) in tibiae from 4-week-old WT and Bmi1 knockout (KO) mice fed with vehicle- or resveratrol (RV)-added diet for 3 weeks before sacrifice. n=6 mice/group. (C) ALP staining on paraffin sections of proximal tibiae. (D) Histomorphometric analysis of ALP + area in total tissue area (ALP + A/T.A). n=6 mice/group. (E) H&E-stained paraffin sections of tibiae and (F) total bone marrow cell numbers in two femora were counted. n=6 mice/group. (G) Cxcl12 gene transcription levels in 3 rd passage of WT and KO BdMPCs treated with vehicle or resveratrol. n=3 samples. (H) ALP staining of 3 rd passage of BdMPCs from 4-week-old WT and KO mice and treated with osteoblast induction medium plus vehicle or resveratrol for 4 days. ALP + cell (red) area was counted. (I) Alp gene transcription levels in bulk mRNA from 3 rd passage of WT and KO BdMPCs treated with vehicle or resveratrol. n=3 samples. (J) Gene transcription levels of niche factors, including Jagged 1 ( Jag1 ), N-cadherin, osteopontin ( Opn ), stem cell factor ( Scf ), Vcam1 , integrin β1 and β2 , in 3 rd passage of WT and KO BdMPCs treated with vehicle or resveratrol tested using qPCR. n=3 samples. *p<0.05, **p<0.01, ***p<0.001. Data are mean ± SD. One-way ANOVA with Tukey's post-hoc test.

Article Snippet: 5×10 6 total bone marrow cells were suspended in 3 ml conditioned culture medium (1.5 ml collected supernatant (serum free) mixed with 1.5 ml fresh culture medium containing 30% FBS) with 20 μg/ml control IgG or 20 μg/ml anti-CXCL12 neutralizing antibody (Millipore; #MABC184) or 20 μg/ml angiopoietin blocking peptide (MyBioSource; #MBS8243845) and seeded in each well of Nunc TM non-treated 6-well plate from Thermo Scientific.

Techniques: Knock-Out, Staining

Journal: International Journal of Biological Sciences

Article Title: Specific overexpression of SIRT1 in mesenchymal stem cells rescues hematopoiesis niche in BMI1 knockout mice through promoting CXCL12 expression

doi: 10.7150/ijbs.63876

Figure Lengend Snippet: Resveratrol dietary supplement partially restored hematopoiesis in Bmi1 KO mice. (A) Representative flow graphs showing gates for LSK cell populations in BM of 4-week-old WT and KO mice that were fed with vehicle- or resveratrol (RV)-added diet for 3 weeks before sacrifice. (B-C) Frequencies (B) and numbers (C) of LSK in total BM cells were counted by flow. (D) Flow gates for LSK cell populations in spleen of mice as in (A) and, (E) the frequencies of LSK cells analyzed by flow. (F) The frequencies of B (B220 + ), T (CD3e + ) and myeloid (CD11b + ) cells in BM of mice in (A) were tested by flow. (G) WB of Sirt1, Cxcl12 and β-actin in protein lysates of WT and KO BdMPCs treated with vehicle or RV for 24 h. (H-J) BM cells from 1-m-old WT mice were cultured in conditioned medium from WT, KO and RV-treated KO BdMPCs plus Ang1 blocking peptide or anti-CXCL12 neutralizing Ab for 48 h. The frequencies of (H) LSK, (I-J) Ki67 + or Annexin V + LSK in culture BM cells were analyzed by flow. n=4 samples. *p<0.05, **p<0.01, ***p<0.001. Data are mean ± SD. One-way ANOVA with Tukey's post-hoc test.

Article Snippet: 5×10 6 total bone marrow cells were suspended in 3 ml conditioned culture medium (1.5 ml collected supernatant (serum free) mixed with 1.5 ml fresh culture medium containing 30% FBS) with 20 μg/ml control IgG or 20 μg/ml anti-CXCL12 neutralizing antibody (Millipore; #MABC184) or 20 μg/ml angiopoietin blocking peptide (MyBioSource; #MBS8243845) and seeded in each well of Nunc TM non-treated 6-well plate from Thermo Scientific.

Techniques: Cell Culture, Blocking Assay

Journal: Theranostics

Article Title: CXCL12-mediated monocyte transmigration into brain perivascular space leads to neuroinflammation and memory deficit in neuropathic pain

doi: 10.7150/thno.44364

Figure Lengend Snippet: SNI produces persistent elevation of CXCL12 in plasma and hippocampal perivascular spaces. ( A ) Cytokine array results showing changes in plasma cytokines and chemokines in sham (Sh) and in 3 d, 9 d after SNI groups. There was no difference in the positive control protein levels (white boxes) among the three groups. ( B, C ) Plasma CXCL12 expression in different groups of female (F) and male (M) mice. n = 3 mice/group. ( D ) ELISA results showing changes in plasma CXCL12 concentrations with time after SNI, compared to sham mice (n = 3-6 mice/group). ( E ) SNI induced upregulation of CXCL12 in bilateral hippocampi (n = 4-6 mice/group). I: ipsilateral hippocampus, C: contralateral hippocampus. ( F ) CXCL12 mRNA in bilateral hippocampi at different time points after SNI (n = 7-12 mice/group). ( G, H ) Expression of CXCL12 around the hippocampal sulcus but not in the CA1 area was upregulated from 1 d to 9 d after SNI compared to sham mice (n = 3-4 slices, 3 mice/group). The white dotted boxes in top images are magnified below. Scale bars: 100 µm (top) and 25 µm (below). n.s.: no significant difference, * P < 0.05, ** P < 0.01, *** P < 0.001 vs. sham group, two-way ANOVA with Bonferroni's post-hoc test used for C and one-way ANOVA with Bonferroni's post-hoc test used for D-G. ( I ) Multiple sets of images show the colocalization of CXCL12 (red) with the PVM marker CD68 (green), endothelial cell marker PECAM-1 (green), astrocyte marker GFAP (green) or pericyte maker CD13 (green) in sham and SNI 3d groups. Red arrows showing the images in I are magnified from the red boxes in H. Scale bar = 25 µm.

Article Snippet: CXCL12 neutralizing antibody (Cat# ab9797, Abcam, Cambridge, UK) was intravenously (i.v.) injected.

Techniques: Positive Control, Expressing, Enzyme-linked Immunosorbent Assay, Marker

Journal: Theranostics

Article Title: CXCL12-mediated monocyte transmigration into brain perivascular space leads to neuroinflammation and memory deficit in neuropathic pain

doi: 10.7150/thno.44364

Figure Lengend Snippet: Blocking the CXCL12-CXCR4 pathway reverses SNI-induced cognitive impairment, PVMs increase, and gliosis in the hippocampus. ( A ) Experimental protocol showing that anti-CXCL12 neutralizing antibody (20 ng/200 µL, i.v.) or CXCR4 antagonist AMD3100 (200 µg/mL, 1 mg/kg, i.p.) or vehicle (Vehi) was applied 30 min before and daily after sham (Sh) or SNI for 9 successive days. On day 9 after the injection, memory function was analyzed with NORT, and mice were perfused for IF and FC. ( B ) Anti-CXCL12 neutralizing antibody or AMD3100 injection prevented SNI-induced decline in the recognition index but had no effect in sham mice (n =5-12 mice/group). ( C-F ) Number of PVMs (CD68 high ) and the IntDen of CD68 high , PECAM-1, CD11b, and GFAP in the hippocampus in indicated groups. Scale bar = 100 µm. n = 3 mice/group, 3-4 slices/mice. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. vehicle sham group, # P < 0.05, ## P < 0.01, ### P < 0.001 vs. vehicle SNI group, one-way ANOVA with Bonferroni's posthoc test.

Article Snippet: CXCL12 neutralizing antibody (Cat# ab9797, Abcam, Cambridge, UK) was intravenously (i.v.) injected.

Techniques: Blocking Assay, Injection

Journal: Theranostics

Article Title: CXCL12-mediated monocyte transmigration into brain perivascular space leads to neuroinflammation and memory deficit in neuropathic pain

doi: 10.7150/thno.44364

Figure Lengend Snippet: Intravenous injection of CXCL12 induces memory deficit and elevates circulating monocytes and plasma CXCL12. ( A ) CXCL12 (1.0 and 2.5 ng/mL, 200 µL) or same volume of vehicle (Vehi) was injected for successive 9 days via the tail vein of naïve mice, and memory function was analyzed by NORT. The blood and brain tissue were harvested for ELISA, FC, and IF after the behavioral test. ( B ) Effects of injection of vehicle or different dosages of CXCL12 on STM index are shown (6-9 mice/group). ( C ) ELISA results revealed that CXCL12 injection at 2.5 ng/mL but not 1.0 ng/mL for 9 days elevated plasma CXCL12 (3-4 mice/group). The plasma CXCL12 was measured 24 h after the last injection of CXCL12. ( D-J ) Effects of iv injection of CXCL12 at 1.0 and 2.5 ng/mL on circulating leukocyte subpopulations. n = 5-6 mice/group. ** P < 0.01, *** P < 0.001 vs. vehicle group, one-way ANOVA with Bonferroni's post hoc test (B, C, H, J) and two-way ANOVA with Bonferroni's post hoc test (E, F).

Article Snippet: CXCL12 neutralizing antibody (Cat# ab9797, Abcam, Cambridge, UK) was intravenously (i.v.) injected.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, IV Injection

Journal: Theranostics

Article Title: CXCL12-mediated monocyte transmigration into brain perivascular space leads to neuroinflammation and memory deficit in neuropathic pain

doi: 10.7150/thno.44364

Figure Lengend Snippet: CXCL12 intravenous injection increases perivascular macrophages and PECAM-1 expression and gliosis in the hippocampus. ( A , B ) Number of PVMs (CD68 high ) and immune fluorescence intensities of CD68 high in the hippocampal perivascular space in vehicle and CXCL12 groups (n = 3 mice, 3-4 slices /mice). ( C, D ) Effect of vehicle or CXCL12 injection on the CD11b, GFAP, PECAM-1, and CXCL12 expression. n = 3-4 slices/mice, 3 mice/group. Scale bar = 100 µm. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. vehicle group, one-way ANOVA with Bonferroni's post-hoc test.

Article Snippet: CXCL12 neutralizing antibody (Cat# ab9797, Abcam, Cambridge, UK) was intravenously (i.v.) injected.

Techniques: Injection, Expressing, Fluorescence

Journal: Theranostics

Article Title: CXCL12-mediated monocyte transmigration into brain perivascular space leads to neuroinflammation and memory deficit in neuropathic pain

doi: 10.7150/thno.44364

Figure Lengend Snippet: Circulating monocytes and plasma CXCL12 are increased in patients with chronic pain and are correlated with cognitive decline. ( A ) Memory function accessed by MoCA in patients with chronic neuropathic pain (Chronic pain, n = 30) was lower than healthy controls (Contr, n = 40). (B-G) Percentages of monocytes ( B ), granulocytes ( C ), neutrophils ( D ), lymphocytes ( E ), eosinophils ( F ), and basophils ( G ) in various groups were determined by routine blood analysis. ( H ) Concentration of plasma CXCL12 in healthy control subjects and chronic pain patients are shown. ** P < 0.01, *** P < 0.001 vs. healthy control group, two-tailed Student's t- test. ( I ) Scatterplots showing that the percentage of circulating monocytes (Spearman rank correlation, R 2 = 0.1022, P = 0.007) and plasma CXCL12 concentration (R 2 = 0.2323, P < 0.0001) was negatively correlated with MoCA scores in healthy controls and patients with chronic pain.

Article Snippet: CXCL12 neutralizing antibody (Cat# ab9797, Abcam, Cambridge, UK) was intravenously (i.v.) injected.

Techniques: Concentration Assay, Two Tailed Test