Journal: The Journal of Experimental Medicine

Article Title: The PSGL-1–L-selectin signaling complex regulates neutrophil adhesion under flow

doi: 10.1084/jem.20130664

Figure Lengend Snippet: The association between PSGL-1 and L-selectin increases after stimulation. (A–C) Representative images of neutrophils rolling on a P-selectin–coated substrate using qDF microscopy. Cells were stained with an antibody for L-selectin (Mel-14, Alexa Fluor 488 coupled; A) or an IgG 2aκ control antibody (Alexa Fluor 488 coupled; B) and DiI for visualizing the cell membrane ( n = 3). The small arrows in A–C signify a ‘sling’ at the front of a rolling neutrophil. Gray arrows indicate the direction of flow. (C) Cells were stained with an Alexa Fluor 488–Mel14 antibody to L-selectin and an Alexa Fluor 568–4RB12 antibody to PSGL-1 showing the distribution of these two molecules on the cell surface during rolling in a flow chamber coated with P-selectin ( n = 3). (D) Quantification of the number of tethers per cell positive for L-selectin or PSGL-1 or showing a colocalization of L-selectin and PSGL-1 ( n = 3). (E and F) Representative images (F) and quantification (E) of the MFI of a PLA of unstimulated and E-selectin–stimulated BM-derived neutrophils from WT mice in the presence or absence of methyl-β-cyclodextrin or its inactive analogue α-cyclodextrin ( n = 3). Using antibodies of different species (4RB12 for PSGL-1 and N-18 for L-selectin) and species-specific secondary antibodies coupled to a DNA strand, a reaction using rolling circle amplification and ligation is started when the two molecules are in close proximity. A fluorescent signal is produced when two tested molecules are within ∼40 nm of each other. DIC, differential interference contrast. (G and H) Quantification of the MFI of FRET experiments comparing unstimulated and E-selectin–stimulated neutrophils. FRET was tested by flow cytometry between L-selectin (labeled by anti–L-selectin antibody LAM1-101 antibody coupled to Alexa Fluor 488) and PSGL-1 (labeled by an anti–PSGL-1 antibody 4RB12 coupled to Alexa Fluor 488) and as a negative control, L-selectin and CXCR2 ( n = 3). (D, E, G, and H) Data are presented as means ± SEM. *, P < 0.05. Bars, 5 µm.

Article Snippet: Tedder, Duke University, Durham, NC), Alexa Fluor 568–conjugated anti–PSGL-1 mAb (clone 4RB12), and/or Alexa Fluor 568–conjugated anti-CXCR2 mAb (R&D Systems).

Techniques: Microscopy, Staining, Membrane, Derivative Assay, Amplification, Ligation, Produced, Flow Cytometry, Labeling, Negative Control

Journal: Molecular Pain

Article Title: Overexpression of GRK6 attenuates neuropathic pain via suppression of CXCR2 in rat dorsal root ganglion

doi: 10.1177/1744806916646381

Figure Lengend Snippet: Reversal of CXCR2 upregulation by GRK6 overexpression in CCI rats. Western blot assays demonstrated a significant upregulation of CXCR1 (a), CXCR2 (b), and CXCR4 (c) protein expression seven days after CCI. * p < .05 vs. Sham. GRK6 overexpression remarkably downregulated CXCR2 protein expression (e) but not CXCR1 (d) and CXCR4 (f) protein expression in CCI rats. * p < .05 vs. NC-LV. CCI: chronic constriction injury; NC-LV: negative control lentivirus.

Article Snippet: The membrane was probed with antibodies against GRK6 and CXCR2 (anti-rabbit, 1:100, Boster, P.R.

Techniques: Over Expression, Western Blot, Expressing, Negative Control

Journal: Molecular Pain

Article Title: Overexpression of GRK6 attenuates neuropathic pain via suppression of CXCR2 in rat dorsal root ganglion

doi: 10.1177/1744806916646381

Figure Lengend Snippet: Correlation of GRK6 and CXCR2 expression and attenuation of neuropathic pain by CXCR2 inhibitor. (a) Expression of CXCR2 protein in DRGs was increased from day 3 to day 21 following CCI. * p < .05 vs. Sham. (b) Correlation analysis showed that the alternations of CXCR2 and GRK6 following CCI were negatively correlated. r 2 = 0.824, p = .042. (c) Co-immunoprecipitation showed the co-localization of CXCR2 and GRK6 in DRGs in CCI rats. (d) Immunofluorescence analysis showed CXCR2 was co-expressed in GRK6 positive DRG neurons. GRK6-positive cells (top left) and β-tubulin-positive cells (bottom left) shown in green. CXCR2-positive cells were shown in red (middle column). Merge of double labeling of GRK6 and CXCR2 (top right) and merge of β-tubulin positive staining and CXCR2 labeling (bottom right). Scale bar = 50 µm. (e, f) Administration of the selective CXCR2 antagonist SB225002 mitigated mechanical hyperalgesia and heat hyperalgesia in CCI rats. Antinociceptive effects of a single intrathecal (it.) injection of 20 µg SB225002 were observed at 1 h and disappeared at 2 h. Antinociceptive effects induced by a single injection of SB225002 at 40 µg were observed at 1 h and disappeared 6 h after injection. Injection of SB225002 (10 µg, i.t.) did not alter the PWT and PWL of CCI rats. N = 6 rats for each group, * p < .05, compared to NS group. PWL: paw withdraw latency; PWT: paw mechanical withdrawal thresholds.

Article Snippet: The membrane was probed with antibodies against GRK6 and CXCR2 (anti-rabbit, 1:100, Boster, P.R.

Techniques: Expressing, Immunoprecipitation, Immunofluorescence, Labeling, Staining, Injection

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