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: Brain

Article Title: Connexin-43 induces chemokine release from spinal cord astrocytes to maintain late-phase neuropathic pain in mice

doi: 10.1093/brain/awu140

Figure Lengend Snippet: Spinal injection of TNF-α-activated astrocytes induces mechanical allodynia via Cx43-mediated CXCL1 release. (A) Intrathecal injection of TNF-α-activated astrocytes elicited persistent mechanical allodynia for >48 h. Note this allodynia is reduced by pretreatment of astrocytes with Cx43 small interfering RNA (1 µg/ml, 18 h). *P < 0.05, compared with TNF-α or TNF-α + non-targeting control small interfering RNA treated group; n = 6 mice/group. (B) ELISA analysis shows increased CXCL1 release in the CSF at 3 h after the intrathecal injection of TNF-α-activated astrocytes. *P < 0.05, compared with vehcile group; #P < 0.05, compared with non-activated astrocytes; n = 4 mice/group. (C) Intrathecal injection of a CXCL1 neutralizing antibody (4 µg) transiently and partially reversed mechanical allodynia, induced by TNF-α-treated astrocytes. *P < 0.05, compared with control IgG group; n = 6 mice/group. (D) Intrathecal injection of the CXCR2 antagonist SB225002 (20 µg = 57 nmol) transiently and partially reversed mechanical allodynia, induced by TNF-α-activated astrocytes. *P < 0.05, compared with vehicle (PBS); n = 5–6 mice/group. All data are mean ± SEM. The differences between groups were analysed by ANOVA followed by Newman–Keuls test.

Article Snippet: The sections were then incubated overnight at 4°C with the following primary antibodies: GFAP antibody (1:1000, mouse; Millipore Bioscience Research Reagents), Cx43 antibody (1:1000, rabbit; Sigma), NeuN antibody (1:1000, mouse; Millipore Bioscience Research Reagents), CXCL1 (1: 200, rabbit; Boster), and CXCR2 antibody (1: 200, rabbit; Boster).

Techniques: Injection, Small Interfering RNA, Control, Enzyme-linked Immunosorbent Assay

Journal: Brain

Article Title: Connexin-43 induces chemokine release from spinal cord astrocytes to maintain late-phase neuropathic pain in mice

doi: 10.1093/brain/awu140

Figure Lengend Snippet: CXCL1, upregulated in spinal cord astrocytes after nerve injury, enhances excitatory synaptic transmission in spinal cord neurons and maintains neuropathic pain via CXCR2. (A) Western blotting shows CXCL1 upregulation in the spinal cord dorsal horn 21 days after CCI. Right, quantification of Cx43 levels in the dorsal horn. The western blot results are presented as a fold of sham control. *P < 0.05, compared to sham control, Student’s t-test, n = 4 mice/group. (B) Intrathecal injection of SB 225002 (20 µg), 21 days after CCI, reduced CCI-induced mechanical allodynia in the late phase. *P < 0.05, compared with vehicle (saline), Student’s t-test, n = 6 mice/group. (C) Double immunostaining of CXCL1 and GFAP in the dorsal horn 21 days after CCI. Note CXCL1 is primarily colocalized with GFAP. Arrows indicate doubled-labelled cells. Scale bar = 50 µm. (D and E) CXCL1 superfusion (100 ng/ml) increases spontaneous EPSC frequency (revealed by patch clamp recordings) in lamina IIo neurons of spinal cord slices. (E) Spontaneous EPSC frequency. *P < 0.05, Student’s t-test, n = 5 neurons/group. (F and G) CCI (21 d) increases spontaneous EPSC frequency, which is reversed by the CXCR2 antagonist SB225002 (1 µM). *P < 0.05, Student’s t-test, n = 5 neurons/group.

Article Snippet: The sections were then incubated overnight at 4°C with the following primary antibodies: GFAP antibody (1:1000, mouse; Millipore Bioscience Research Reagents), Cx43 antibody (1:1000, rabbit; Sigma), NeuN antibody (1:1000, mouse; Millipore Bioscience Research Reagents), CXCL1 (1: 200, rabbit; Boster), and CXCR2 antibody (1: 200, rabbit; Boster).

Techniques: Transmission Assay, Western Blot, Control, Injection, Saline, Double Immunostaining, Patch Clamp

Journal: Brain

Article Title: Connexin-43 induces chemokine release from spinal cord astrocytes to maintain late-phase neuropathic pain in mice

doi: 10.1093/brain/awu140

Figure Lengend Snippet: Schematic of working hypothesis for astrocytic Cx43-mediated late-phase neuropathic pain. CCI induces a persistent upregulation of Cx43 in spinal cord astrocytes. Cx43 expression and activity is also upregulated by TNF-α, secreted from microglia. Upregulation of Cx43 hemichannel activities results in CXCL1 release. Astrocytic CXCL1 secretion activates CXCR2 on neurons (central terminals of primary sensory neurons and spinal cord neurons), leading to enhanced excitatory synaptic transmission in nociceptive neurons (e.g. lamina IIo excitatory interneurons) and sustained neuropathic pain in the late-phase. Additionally, CXCL1 can also be secreted from intact or injured primary afferents in the spinal cord especially in the early phase of CCI.

Article Snippet: The sections were then incubated overnight at 4°C with the following primary antibodies: GFAP antibody (1:1000, mouse; Millipore Bioscience Research Reagents), Cx43 antibody (1:1000, rabbit; Sigma), NeuN antibody (1:1000, mouse; Millipore Bioscience Research Reagents), CXCL1 (1: 200, rabbit; Boster), and CXCR2 antibody (1: 200, rabbit; Boster).

Techniques: Expressing, Activity Assay, Transmission Assay

Journal: Lung cancer (Amsterdam, Netherlands)

Article Title: Combination therapy with anti-programmed cell death 1 antibody plus angiokinase inhibitor exerts synergistic antitumor effect against malignant mesothelioma via tumor microenvironment modulation.

doi: 10.1016/j.lungcan.2023.107219

Figure Lengend Snippet: Fig. 1. MPM cells secreted multiple angiogenic factors. A, Comprehensive analysis of the expression of transcripts of multiple angiogenic factors (VEGFA, PDGFb, FGF2, HGF, ANG1 and IL-8) in human umbilical vein endothelial cell (HUVEC), mesothelial cell (Met-5A) and MPM cell lines (MSTO-211H, H2452, H2052, and H28) by qRT-PCR. Expression of FGF2 transcripts in all MPM cells was higher than in HUVECs. B, Comprehensive analysis of the protein expression of angiogenic factor receptors (VEGFR2, PDGFRβ, FGFR1, c-Met, Tie2 and CXCR1) in HUVEC, human mesothelial cell and MPM cells by immunoblotting. MPM cells expressed higher level of FGFR1 compared with HUVECs. C, Antiproliferative effect of multi-angiokinase inhibitor nintedanib (NTD) in MPM cells. Mesothelial cells and MPM cells were treated with serially diluted NTD for up to 72 h. The relative numbers of viable cells were quantified using CCK-8 assay. Points, mean % viable cells; bars, SEM of at least three independent experiments performed in triplicate.

Article Snippet: Rabbit polyclonal Ab against CXC motif chemokine receptor 1 (CXCR1) (#55450–1- AP) was obtained from Proteintech (Rosemont, IL).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, CCK-8 Assay