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: Nature Communications

Article Title: An isoform quantitative trait locus in SBNO2 links genetic susceptibility to Crohn’s disease with defective antimicrobial activity

doi: 10.1038/s41467-024-47218-3

Figure Lengend Snippet: a Volcano plots illustrate differential gene expression following siRNA-mediated SBNO2 knockdown in unstimulated MDM and MDM following 8 h stimulation with IL-10, LPS, or LPS + aIL-10R analysed by RNA-seq and DESeq2 ( n = 3, log2 fold change (log 2 fc) > 0.5, p adj < 0.05, FDR). b RT-qPCR validation of target genes of interest found differentially regulated by SBNO2 knockdown in RNA-seq experiments (Independent experiments/donors: IL23A : n = 6/12; IL20, IL24 : n = 3/7; CXCR2, CXCR4, KREMEN1 : n = 4/9; non-parametric, two-sided, Friedman test). c The bar graph illustrates the overlap of functional pathways (GO Biologic Processes, KEGG pathways, and Reactome Pathways) based on STRING database functional enrichment analysis of SBNO2 knockdown differentially regulated genes in LPS- and LPS + aIL-10R-stimulated MDM. The total number of enriched pathways is indicated. d – f The heatmap shows the enrichment score of STRING database functional enrichment analysis showing the ( e ) top 10 downregulated/SBNO2-induced pathways, ( f ) top 10 upregulated/SBNO2-suppressed pathways, and top 10 enriched pathways without polarity in the dataset based on DESeq2 log 2 fold change differential expression ranking. g Examples of enriched functional pathway terms and list of respective top 5 SBNO2-regulated pathway genes (enrichment score). The distribution according to DESeq2 log 2 fold change expression across the data are visualized. Source data are provided as a Source Data file for Fig. 4b.

Article Snippet: The following TaqMan probes (Applied Biosystems) were used for qPCR analysis: RPLPO (Hs99999902_m1), SBNO2 (ISO1/ISO2) (Hs00922127_m1), SBNO2 (ISO1) (Hs00209130_m1), IL20 (Hs00218888_m1), IL23A (Hs00900828_g1), IL24 (Hs01114274_m1), CXCR2 (Hs00174304_m1), CXCR4 (Hs00976734_m1), KREMEN1 (Hs00230750_m1)

Techniques: Gene Expression, Knockdown, RNA Sequencing, Quantitative RT-PCR, Biomarker Discovery, Functional Assay, Quantitative Proteomics, Expressing

Journal: Journal of Neuroinflammation

Article Title: Glial remodeling enhances short-term memory performance in Wistar rats

doi: 10.1186/s12974-020-1729-4

Figure Lengend Snippet: TaqMan probe details (Life Technologies) used for qrt-PCR

Article Snippet: Cxcr2 , NM_017183.1 , Rn02130551_s1 , 122.

Techniques: Sequencing, TaqMan Assay

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