Journal: British Journal of Cancer

Article Title: Early expression of the fractalkine receptor CX3CR1 in pancreatic carcinogenesis

doi: 10.1038/bjc.2013.565

Figure Lengend Snippet: CX3CR1 and CX3CL1 expression in normal pancreas and in pancreatic cancer. ( A ) CX3CR1 expression in normal pancreas, ( B ) in well- and ( C ) in poorly differentiated PDAC, and ( B , inset) in inflammatory cells in tumour microenvironment. ( D ) CX3CL1 expression in normal pancreas, ( E ) in neural cell bodies, and ( F ) in PDAC (box, × 40 magnification of a CX3CL1 + PDAC). Objective magnification × 10 ( A – D and F ); ( E ) and inset in ( B ), × 40. ( G ) Frequency of CX3CR1 + and CX3CR1 − PDAC with PNI according to tumour differentiation (Grade, G1–G2 well-to-moderately differentiated PDAC; G3, poorly differentiated PDAC). ( H ) Kaplan–Meier curves for overall survival of patients after radical resection of PDAC ( n =67), according to the status of CX3-CR1 (left panel) and -CL1 (right panel) in cancer. P -values by log-rank test. * indicates P <0.05.

Article Snippet: After deparaffining and rehydration, the sections were immersed in antigen retrieval bath, incubated with 3% H 2 O 2 for 15 min, and treated for 2 h at room temperature with primary antibodies raised against CX3CR1 (Ab8021, polyclonal rabbit anti-human; Abcam, Cambridge, UK) and CX3CL1 (AF365, polyclonal goat anti-human; R&D Systems, Minneapolis, MN, USA), or with rabbit or goat IgG (Dako, Milan, Italy) to serve as negative controls, followed by 30-min incubation with the DAKO Envision system (Dako) or the Anti-Goat Polymer kit (Biocare, San Francisco, CA, USA).

Techniques: Expressing

Journal: British Journal of Cancer

Article Title: Early expression of the fractalkine receptor CX3CR1 in pancreatic carcinogenesis

doi: 10.1038/bjc.2013.565

Figure Lengend Snippet: CX3CR1 and CX3CL1 expression in 104 PDAC according to patients' demographics and to tumour features

Article Snippet: After deparaffining and rehydration, the sections were immersed in antigen retrieval bath, incubated with 3% H 2 O 2 for 15 min, and treated for 2 h at room temperature with primary antibodies raised against CX3CR1 (Ab8021, polyclonal rabbit anti-human; Abcam, Cambridge, UK) and CX3CL1 (AF365, polyclonal goat anti-human; R&D Systems, Minneapolis, MN, USA), or with rabbit or goat IgG (Dako, Milan, Italy) to serve as negative controls, followed by 30-min incubation with the DAKO Envision system (Dako) or the Anti-Goat Polymer kit (Biocare, San Francisco, CA, USA).

Techniques: Expressing

Journal: British Journal of Cancer

Article Title: Early expression of the fractalkine receptor CX3CR1 in pancreatic carcinogenesis

doi: 10.1038/bjc.2013.565

Figure Lengend Snippet: Tumour differentiation, peri-neural invasion, and ligand expression are independently associated with CX3CR1 expression

Article Snippet: After deparaffining and rehydration, the sections were immersed in antigen retrieval bath, incubated with 3% H 2 O 2 for 15 min, and treated for 2 h at room temperature with primary antibodies raised against CX3CR1 (Ab8021, polyclonal rabbit anti-human; Abcam, Cambridge, UK) and CX3CL1 (AF365, polyclonal goat anti-human; R&D Systems, Minneapolis, MN, USA), or with rabbit or goat IgG (Dako, Milan, Italy) to serve as negative controls, followed by 30-min incubation with the DAKO Envision system (Dako) or the Anti-Goat Polymer kit (Biocare, San Francisco, CA, USA).

Techniques: Expressing

Journal: British Journal of Cancer

Article Title: Early expression of the fractalkine receptor CX3CR1 in pancreatic carcinogenesis

doi: 10.1038/bjc.2013.565

Figure Lengend Snippet: Expression of CX3CR1 and CX3CL1 in precursor lesions according to tumour differentiation and PanIN degree. ( A ) Comparison of the expression rate of CX3CR1 (upper panels) and CX3CL1 (lower panels) in paired precursor lesions and invasive cancers within the same tissue specimens ( n =76), according to the degree of pancreatic cancer differentiation. Number within bars, number of specimens. ( B ) Rates of CX3CR1 + (upper panels) and CX3CL1 + (lower panels) PanINs according to their degree of dysplasia. Numbers within bars, number of PanINs. G1–G2 tumours, left panels; G3 tumours, right panels; P -values by Fisher's exact test.

Article Snippet: After deparaffining and rehydration, the sections were immersed in antigen retrieval bath, incubated with 3% H 2 O 2 for 15 min, and treated for 2 h at room temperature with primary antibodies raised against CX3CR1 (Ab8021, polyclonal rabbit anti-human; Abcam, Cambridge, UK) and CX3CL1 (AF365, polyclonal goat anti-human; R&D Systems, Minneapolis, MN, USA), or with rabbit or goat IgG (Dako, Milan, Italy) to serve as negative controls, followed by 30-min incubation with the DAKO Envision system (Dako) or the Anti-Goat Polymer kit (Biocare, San Francisco, CA, USA).

Techniques: Expressing, Comparison

Journal: British Journal of Cancer

Article Title: Early expression of the fractalkine receptor CX3CR1 in pancreatic carcinogenesis

doi: 10.1038/bjc.2013.565

Figure Lengend Snippet: CX3CR1 and CX3CL1 expression in precursor lesions of pancreatic cancer. ( A ) Left panels, human PanINs; right panels, PanINs of PdxCre/LSL- Kras G12D mice. Objective magnification, × 20 (human PanINs), and × 40 (mice PanINs). ( B ) Precursor lesions adjacent to neural structures. Detection of precursor lesions close to (left panel) or in tight contact with (right panel) S-100 + neural structures (red arrows) in pancreata of 6-month-old PdxCre/LSL-Kras G12D mice. Magnification, × 20.

Article Snippet: After deparaffining and rehydration, the sections were immersed in antigen retrieval bath, incubated with 3% H 2 O 2 for 15 min, and treated for 2 h at room temperature with primary antibodies raised against CX3CR1 (Ab8021, polyclonal rabbit anti-human; Abcam, Cambridge, UK) and CX3CL1 (AF365, polyclonal goat anti-human; R&D Systems, Minneapolis, MN, USA), or with rabbit or goat IgG (Dako, Milan, Italy) to serve as negative controls, followed by 30-min incubation with the DAKO Envision system (Dako) or the Anti-Goat Polymer kit (Biocare, San Francisco, CA, USA).

Techniques: Expressing

Journal: British Journal of Cancer

Article Title: Early expression of the fractalkine receptor CX3CR1 in pancreatic carcinogenesis

doi: 10.1038/bjc.2013.565

Figure Lengend Snippet: Effects of microenvironmental stimuli on CX3CR1 and CX3CL1 expression in pancreatic tumoral cells. CX3CR1 (left panels) and CX3CL1 (right panels) mRNA fold changes induced by stimulation with TNF- α +IFN- γ , IL-1 β , IL-6, and by TGF- β in mouse DT6606 ( PdxCre/LSL-Kras G12 model) and K8484 ( PdxCre/LSL - Kras G12D - Trp53 R172H model) cells, and in human A8184, AspCI, and MiaPacaII pancreatic cancer cells. * indicates P <0.05.

Article Snippet: After deparaffining and rehydration, the sections were immersed in antigen retrieval bath, incubated with 3% H 2 O 2 for 15 min, and treated for 2 h at room temperature with primary antibodies raised against CX3CR1 (Ab8021, polyclonal rabbit anti-human; Abcam, Cambridge, UK) and CX3CL1 (AF365, polyclonal goat anti-human; R&D Systems, Minneapolis, MN, USA), or with rabbit or goat IgG (Dako, Milan, Italy) to serve as negative controls, followed by 30-min incubation with the DAKO Envision system (Dako) or the Anti-Goat Polymer kit (Biocare, San Francisco, CA, USA).

Techniques: Expressing

Journal: Physiological Reports

Article Title: Tuberous sclerosis complex exhibits a new renal cystogenic mechanism

doi: 10.14814/phy2.13983

Figure Lengend Snippet: List of antibodies used in the study

Article Snippet: Rabbit polyclonal Anti‐NCC , 1:60 , Stressmarq Biosciences; Victoria, BC, Canada , SPC‐402.

Techniques: Plasmid Preparation, Generated

Journal: Journal of immunology research

Article Title: Diet-Induced Obesity Promotes Liver Metastasis of Pancreatic Ductal Adenocarcinoma via CX3CL1/CX3CR1 Axis.

doi: 10.1155/2022/5665964

Figure Lengend Snippet: Figure 4: DIO increased expression level of CX3CL1 and CX3CR1 in liver metastasis of PDAC. (a) Relative mRNA levels of CX3CL1 in liver of ND mice and DIO mice after intrasplenic injection with 1 × 106 Panc02 cells (n = 6/group). (b) Representative immunohistochemical images of CX3CL1 staining in liver of ND and DIO. Scale bar, 20 μm. (c) Bar graph showed the relative CX3CL1 staining positive rate. (d) Relative mRNA levels of CX3CL1 in liver of ND mice and DIO mice after intrasplenic injection with 1 × 106

Article Snippet: The cells were incubated with CX3CR1 primary antibody (1 : 100, Proteintech,13885-1-AP) for 30min in the dark at 4°C.

Techniques: Expressing, Injection, Immunohistochemical staining, Staining

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: Cx43 is required for TNF-α-evoked and basal release of CXCL1 in astrocyte cultures. (A and B) CXCL1 release in astrocytes following TNF-α stimulation (10 ng/ml, 60 min). Note the TNF-α-induced CXCL1 release is suppressed by pretreatment (60 min) of CBX (20 and 100 µM, A) and Gap26 and Gap27 (100 µM, B) but not by the inhibitors of pannexin hemichannels probenecid (Prob, 500 µM, A) and PANX1 mimetic peptide 10Panx1 (100 µM, A) and the scrambled peptide (Gap27 scrambled, 100 µM, B). *P < 0.05, compared with control; #P < 0.05, compared with TNF-α. (C) Inhibition of basal release of CXCL1 by CBX in astrocytes. *P < 0.05, compared with control. (D) Evoked expression (content) of CXCL1 in astrocytes following TNF-α stimulation (10 ng/ml, 60 min). Note the TNF-α-induced CXCL1 expression is not suppressed by pretreatment (60 min) of CBX (20 and 100 µM) and inhibitors of pannexin hemichannels probenecid (Prob, 500 µM) and PANX1 mimetic peptide 10Panx1 (100 µM). In contrast, a high dose of CBX (100 µM) increases CXCL1 expression. *P < 0.05, compared with control; #P < 0.05, compared with TNF-α. (E) Effects of CBX on the basal expression (content) of CXCL1 in astrocytes. All data are mean ± SEM. n = 8 cultures/group. 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: Control, Inhibition, 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