Journal: bioRxiv

Article Title: Mechanistic insights of radiation-induced endothelial senescence impelling glioblastoma genomic instability at relapse

doi: 10.1101/2021.12.13.472364

Figure Lengend Snippet: a. CXCR2 expression in U251 and human primary GBM cells by FACS analysis. Left panel : Representative histograms of CXCR2 staining in U251 cells. Right panel : Geomean of fluorescence compared to isotype. Geomean of CXCR2 expression in U251 cells and GBM primary cells is indicated respectively as black and white circles. b. Radio-induced MN production per cell after 5Gy in U251 cells in CM or SASP after CXCR2 blocking using monoclonal antibody MAb331(250ng/ml) or SB332235 antagonist (1nM). (mean±SD, n=3, one-way ANOVA; * p<0.05, *** p<0.001). c. Polynucleated cell frequency after 5Gy-radiation in U251 cells in CM or SASP after CXCR2 blocking using monoclonal antibody MAb331(250ng/ml) or SB332235 antagonist (1nM). (mean±SD; n=3; two-way ANOVA, * p<0.05, ** p<0.01). d. Elongation rate of U251 cells in CM (black) or SASP (blue) supplemented or not with CXCR2 monoclonal antibody (MAb331, 250ng/ml). When indicated, cells were irradiated with 5Gy-radiation. (mean±SD, n=3, two-way ANOVA; ** p<0.01, *** p<0.001).

Article Snippet: Anti-CXCR2 blocking antibody (Mab331) and antagonist drug (SB332235) were purchased from R&D and TOCRIS (Bioscience) respectively.

Techniques: Expressing, Staining, Fluorescence, Blocking Assay, Irradiation

Journal: bioRxiv

Article Title: Mechanistic insights of radiation-induced endothelial senescence impelling glioblastoma genomic instability at relapse

doi: 10.1101/2021.12.13.472364

Figure Lengend Snippet: a. Median survival (days) of tumor-bearing mice after orthotopic injections of radiation-surviving U251 cells obtained in presence of CM (R15CM), SASP (R15SASP), CM supplemented with CXCL8 and CXCL5 (R15CM -CXCL5/8 ), SASP supplemented with either CXCR2 monoclonal antibody Mab331 (R15SASP- Mab ) or CXCR2 antagonist SB3322235 (R15SASP- SB ). Log-rank p-values are indicated as compare to mice survival injected with either R15CM radioresistant cells or R15SASP radioresistant cells. b. Tumor-bearing mice survival after orthotopic injections of R15CM-CXCL (left panel), R15SASP-mAbR2 (middle panel) and R15SASP-SBR2 (right panel) (n=8/group) as compare to R15CM and R15SASP tumor-bearing mice. c. Immunofluorescence of CXCL5 and CXCL8 in primary and recurrent human GBM. d. Kaplan-Meier plots overall survival of GBM patients depending on CXCL5 and CXCL8 mRNA expression using the TCGA-glioblastoma database. (Log-rank tests analysis).

Article Snippet: Anti-CXCR2 blocking antibody (Mab331) and antagonist drug (SB332235) were purchased from R&D and TOCRIS (Bioscience) respectively.

Techniques: Injection, Immunofluorescence, Expressing

Journal: Journal of translational medicine

Article Title: RBM15 recruits myeloid-derived suppressor cells via the m6A-IGF2BP3/CBR3-AS1/miR-409-3p/CXCL1 axis, facilitating radioresistance in non-small-cell lung cancer.

doi: 10.1186/s12967-025-06205-y

Figure Lengend Snippet: Fig. 4 CBR3-AS1 recruits MDSCs through the miR-409-3p/CXCL1/CXCR2 axis. A Relative mRNA expression levels of CXCL1 in H520 cells transfected with a control empty vector (vecCtrl), miR-409-3p mimic plasmid (miR-409-3p), control shRNA (shCtrl), or specific interfering hairpin RNAs targeting miR-409-3p (shmiR-409-3p). The cells were exposed to radiation after 24 h of transfection. B Relative protein expression levels of CXCL1 in H520 cells. β-actin was used as the reference for normalization. C Relative luciferase activity of CXCL1 under miR-409-3p overexpression in the wild-type and mutant CXCL1 groups treated with radiation. D, E Relative mRNA and protein expression levels of CXCL1 in H520 cells transfected with shCtrl, shCBR3-AS1, or shmiR-409-3p. F Migration ability of MDSCs toward the conditioned medium of H520 cells transfected with CXCL1, shCXCL1, or treated with a CXCR2 inhibitor (SB265610, 10 mM) and treated with radiation. G, H Levels of interferon (IFN)-γ secreted by CD8+ and CD4.+ T cells co-cultured with H520 cells transfected with CXCL1, shCXCL1 or treated with a CXCR2 inhibitor (SB265610, 10 mM). IFN-γ expression was reported as the mean fluorescence intensity. The significance between two groups was analyzed using Student’s t-test. Data are presented as mean ± SD from three independent experiments. Statistical significance was defined as *P < 0.05, **P < 0.01, and ***P < 0.001

Article Snippet: Membranes were probed with primary antibodies: RBM15 (1:500, Abclonal, USA, A4936), IGF2BP3 (1:1000, Abclonal, A4444), CXCL1 (1:500, Abclonal, USA, A5802), CXCR2 (1:1000, Proteintech, Wuhan, China, 20634-1-AP), or β-actin (1:500, Abclonal, USA, WL01372).

Techniques: Expressing, Transfection, Control, Plasmid Preparation, shRNA, Luciferase, Activity Assay, Over Expression, Mutagenesis, Migration, Cell Culture, Fluorescence

Journal: Mucosal Immunology

Article Title: Butyrate regulates neutrophil homeostasis and impairs early antimicrobial activity in the lung

doi: 10.1016/j.mucimm.2023.05.005

Figure Lengend Snippet: Effect of butyrate on acute Pseudomonas aeruginosa immunity. (A) Quantification of the total number of cells in BALF 18 hours post-inoculation with 1 × 10 6 CFU of P. aeruginosa strain PAO1 in Ctrl and But mice. (B) Frequency and quantification of cell types in cytospins of BALF from control and butyrate-treated mice 18 hours after PAO1 infection; (C) Quantification of P. aeruginosa CFU in BALF 18 hours post-challenge. (D, E) Cytokine and chemokine production in BALF 18 hours following infection. (F) MFI of CXCR2 on lung AM and IM from Ctrl and But mice. (G) MFI of CXCR2 on Ctrl and But lung macrophages US or stimulated for 2 hours with LPS in vitro . (H) transmigration of Ctrl and But neutrophils toward a CXCL2 or CCL2 chemokine gradient after 3 hours. Results are a mean of two independent experiments. Values are expressed as mean ± standard error of mean; n = 6–14. Statistical significance was determined with one-way analysis of variance in (A–E, G, H) and Student’s t test (unpaired, two-tailed) in (F). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. AM = alveolar macrophages; BALF = bronchoalveolar lavage fluid; But = butyrate-treated; CFU = colony-forming units; Ctrl = control; IFN = interferon; IL = interleukin; IM = interstitial macrophages; LPS = lipopolysaccharide; MFI = mean fluorescence intensity; PAO1 = P. aeruginosa strain 1; TNF = tumor necrosis factor; US = unstimulated.

Article Snippet: To differentiate progenitor and neutrophil populations in the BM, cells were stained with antibodies to lineage cocktail, CD115 (BD Biosciences, clone 2B8), SiglecF (BD Biosciences, clone 2B8), CD117 BUV117 (BD Biosciences, clone 2B8), Sca1 APCFire (Biolegend, clone D7), FcgR biot (Miltenyi, clone 93), CD34 BV421 (Biolegend, clone SA376A4), CD11b BV510 (BD Biosciences, clone M1/70), Ly6G Pecy5 (eBioscience, clone 1A8-Ly6g), CXCR2 Pecy7 (Miltenyi, clone REA942) and streptavidin BUV395 (BD Biosciences).

Techniques: Control, Infection, In Vitro, Transmigration Assay, Two Tailed Test, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: The Chemokine Receptor CCR3 Is Potentially Involved in the Homing of Prostate Cancer Cells to Bone: Implication of Bone-Marrow Adipocytes

doi: 10.3390/ijms22041994

Figure Lengend Snippet: CCR3 is an important driver in the directed migration of PCa cells towards BM-Ad-CM and its effect is majored in obesity and ageing conditions. In vitro migration of C4-2B ( A ) or PC3 ( B ) . CCR3 (UCB35625, 200 nM), CCR2 (sc-202525, 25 nM), CXCR1/2 (SB225002 inhibitor, 50 nM) or CXCR4 (AMD3100, 100 nM) or with mAbs against CCR3, CXCR1, CXCR2 or CXCR4 (all used at 10 μg/ml). Bar plots represent the percentage of migrating cells relative to the migration of untreated cells (set to 100%). Data are shown as mean ± sem ( n = 4–6). The statistical significance between means of migrating cells (in %) in treated vs. control cells was evaluated by One-way ANOVA with Tukey’s multiple comparisons test. Similar experiments were performed with the indicated cell lines towards BM-Ad-CM obtained either from lean/obese ( C ) or control/aged ( D ) subjects in the presence of CCR3 inhibitors and blocking mAb. Data are shown as mean ± sem ( n = 4–9). The statistical significance between mean of migrating cells (with the control groups set at 100%) was evaluated by Two-way ANOVA with Sidak’s multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant.

Article Snippet: When indicated, cells were pre-incubated for 30 min at 37 °C with 10 μg/mL blocking monoclonal antibodies (mAbs) directed against CCR3 (clone 444-11, reference D085-3, obtained from MBL International, Woburn, MA, USA), CXCR1 (Clone 42705, reference MAB330), CXCR2 (clone 48311, reference MAB331), CXCR4 (clone 44716, reference MAB172) or control IgG (all obtained from R&D Systems, Minneapolis, MN, USA).

Techniques: Migration, In Vitro, Control, Blocking Assay

Journal: International Journal of Molecular Sciences

Article Title: The Chemokine Receptor CCR3 Is Potentially Involved in the Homing of Prostate Cancer Cells to Bone: Implication of Bone-Marrow Adipocytes

doi: 10.3390/ijms22041994

Figure Lengend Snippet: Expression of chemokine receptors in primary versus bone metastases of human PCa. The cancer microarray database, Oncomine was used to compare the RNA expression of several chemokine receptors (CCR2, CCR3, CXCR2 and CXCR4) in patient samples collected from bone metastatic (grey squares) or primary sites (white squares) from six different data-sets. Differences between the two independent groups were evaluated with Mann-Whitney’s test. *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: When indicated, cells were pre-incubated for 30 min at 37 °C with 10 μg/mL blocking monoclonal antibodies (mAbs) directed against CCR3 (clone 444-11, reference D085-3, obtained from MBL International, Woburn, MA, USA), CXCR1 (Clone 42705, reference MAB330), CXCR2 (clone 48311, reference MAB331), CXCR4 (clone 44716, reference MAB172) or control IgG (all obtained from R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Microarray, RNA Expression