ccr5 blockage  (TargetMol)

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Chemotherapy-elicited extracellular vesicle CXCL1 from dying cells promotes triple-negative breast cancer metastasis by activating TAM/PD-L1 signaling.

doi: 10.1186/s13046-024-03050-7

Figure Lengend Snippet: Fig. 4 CXCL1EV-dead induces macrophage M2 polarization by activating PD-L1 expression. A Chemokine array assay was conducted to characterize the differences in chemokine content between EV-dead and EV-alive. An ELISA assay was conducted to compare the relative CXCL1 content in EV-dead and EV-alive. B Changes in M2 phenotype polarization of Raw264.7 macrophages when treated with 10 ng/ml murine CXCL1, 50 μg/ ml EV-dead, 50 μg/ml EV-deadshCXCL1, 5 μg/ml CXCL1 neutralizing antibody (NA), or EV-dead and CXCL1-NA combination for 48 h. C Representative images of Transwell assay. Raw264.7 macrophages were treated as indicated for 48 h and then co-cultured with 4 T1 cells. Scale bar: 200 μm. D–F Expression changes of CXCL1 and PD-L1 in Raw264.7 macrophages when treated as indicated for 48 h. Scale bar: 10 μm. G The results of the flow cytometry assay suggested that 50 μg/ml EV-dead treatment for 48 h induced the M2 polarization of Raw264.7 macrophages by activating PD-L1 expression; n = 3. Data are presented as mean ± SD. *p < 0.05, **p < 0.01

Article Snippet: CXCL1 secretion inhibitor screening To screen the potential CXCL1 secretion inhibitor of 4 T1 cells from the Chemokine Inhibitor Library (L7600, TOPSCIENCE, Shanghai, China), 4 T1 cells were treated with 80 types of chemokine inhibitors (1 μM) for 48 h. Subsequently, the concentration of CXCL1 in cell culture supernatants was detected using the Mouse CXCL1 ELISA Kit.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Transwell Assay, Cell Culture, Flow Cytometry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chemotherapy-elicited extracellular vesicle CXCL1 from dying cells promotes triple-negative breast cancer metastasis by activating TAM/PD-L1 signaling

doi: 10.1186/s13046-024-03050-7

Figure Lengend Snippet: CXCL1 EV-dead induces macrophage M2 polarization by activating PD-L1 expression. A Chemokine array assay was conducted to characterize the differences in chemokine content between EV-dead and EV-alive. An ELISA assay was conducted to compare the relative CXCL1 content in EV-dead and EV-alive. B Changes in M2 phenotype polarization of Raw264.7 macrophages when treated with 10 ng/ml murine CXCL1, 50 μg/ml EV-dead, 50 μg/ml EV-dead shCXCL1 , 5 μg/ml CXCL1 neutralizing antibody (NA), or EV-dead and CXCL1-NA combination for 48 h. C Representative images of Transwell assay. Raw264.7 macrophages were treated as indicated for 48 h and then co-cultured with 4 T1 cells. Scale bar: 200 μm. D–F Expression changes of CXCL1 and PD-L1 in Raw264.7 macrophages when treated as indicated for 48 h. Scale bar: 10 μm. G The results of the flow cytometry assay suggested that 50 μg/ml EV-dead treatment for 48 h induced the M2 polarization of Raw264.7 macrophages by activating PD-L1 expression; n = 3. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01

Article Snippet: To screen the potential CXCL1 secretion inhibitor of 4 T1 cells from the Chemokine Inhibitor Library (L7600, TOPSCIENCE, Shanghai, China), 4 T1 cells were treated with 80 types of chemokine inhibitors (1 μM) for 48 h. Subsequently, the concentration of CXCL1 in cell culture supernatants was detected using the Mouse CXCL1 ELISA Kit.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Transwell Assay, Cell Culture, Flow Cytometry

Journal: Journal of Neuroinflammation

Article Title: CXCL12 is involved in α-synuclein-triggered neuroinflammation of Parkinson’s disease

doi: 10.1186/s12974-019-1646-6

Figure Lengend Snippet: The levels of CXCL12 and CXCR4 were elevated in the brain tissues of PD patients and A53T transgenic mice. a Western blot analysis showed the expression of CXCL12 in SN sections of postmortem brain tissue from control and PD subjects. GAPDH was used as an internal control for gel loading, and M17 cell protein was loaded as a positive control. b Western blot analysis showed an upregulation of CXCR4 and pser129 a-syn protein levels in human PD brains compared with the control. c The mRNA expression of CXCL12 and CXCR4 was determined by real-time qPCR in A53T and WT mice. n = 3 per group. d Brain slices from the substantia nigra were stained for CXCL12 and CXCR4 by immunohistochemistry. n = 3 per group for A53T and WT mice. e Brain slices from the substantia nigra were double stained for IBA-1 (red) and CXCR4 (green). Images were captured with a fluorescence microscope. Scale bar = 100 μm. n = 3 per group. f Brain tissue from the substantia nigra was stained for microglia (CD11b-FITC) and CXCR4 (APC), and the frequency of the CD11b+ and CD11b+CXCR4+ cells was assessed by flow cytometry. n = 3 per group for A53T and WT mice. The data are shown as the mean ± SEM for three independent experiments. ** p < 0.01. * p < 0.05

Article Snippet: AMD3100 (1 ng/ml, TargetMol, USA) was used as a CXCR4 antagonist, TAK242 (Merck, Germany) was used as a TLR4 inhibitor at a concentration of 100 nM, pyrrolidine dithiocarbamate (PDTC, 50 μM, TargetMol) was applied as an inhibitor of NF-κb (nuclear factor kappa), and C29 was used as a blocker of TLR2 signaling (50 μM, TargetMol).

Techniques: Transgenic Assay, Western Blot, Expressing, Control, Positive Control, Staining, Immunohistochemistry, Fluorescence, Microscopy, Flow Cytometry

Journal: Journal of Neuroinflammation

Article Title: CXCL12 is involved in α-synuclein-triggered neuroinflammation of Parkinson’s disease

doi: 10.1186/s12974-019-1646-6

Figure Lengend Snippet: α-Synuclein upregulated the expression of CXCL12 in microglia. a Primary microglia were sorted by anti-CD11b-conjugated microbeads from 12 neonatal mice once. b The sorting efficiency was assessed by flow cytometry. c The concentration of CXCL12 in the supernatant of primary microglia was measured by ELISA after stimulation by α-synuclein or BSA. d BV-2 cells were stimulated by α-synuclein or BSA; supernatants were collected at 6, 12, 24, and 48 h; and the concentration of CXCL12 was evaluated by ELISA. e The expression of CXCR4 in BV-2 cells was determined by western blot after stimulation by α-synuclein. f BV-2 cells were stained for CXCR4 (red) after stimulation for 48 h with α-synuclein. Images were captured with a fluorescence microscope. Scale bar = 25 μm. The data are shown as the mean ± SEM for three independent experiments. ** p < 0.01. * p < 0.05. BSA served as a control

Article Snippet: AMD3100 (1 ng/ml, TargetMol, USA) was used as a CXCR4 antagonist, TAK242 (Merck, Germany) was used as a TLR4 inhibitor at a concentration of 100 nM, pyrrolidine dithiocarbamate (PDTC, 50 μM, TargetMol) was applied as an inhibitor of NF-κb (nuclear factor kappa), and C29 was used as a blocker of TLR2 signaling (50 μM, TargetMol).

Techniques: Expressing, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Staining, Fluorescence, Microscopy, Control

Journal: Journal of Neuroinflammation

Article Title: CXCL12 is involved in α-synuclein-triggered neuroinflammation of Parkinson’s disease

doi: 10.1186/s12974-019-1646-6

Figure Lengend Snippet: CXCL12/CXCR4 was involved in α-synuclein-induced microglial migration. a The migration of BV-2 cells towards CXCL12 was measured by Transwell assay, and AMD3100 was added to the upper chambers to block the interaction between CXCL12 and CXCR4. b The migration of BV-2 cells induced by α-synuclein was observed with or without pretreatment with a TLR4 inhibitor (TAK242) or an NF-κB inhibitor (PDTC). The data are shown as the mean ± SEM from three independent experiments. * p < 0.05

Article Snippet: AMD3100 (1 ng/ml, TargetMol, USA) was used as a CXCR4 antagonist, TAK242 (Merck, Germany) was used as a TLR4 inhibitor at a concentration of 100 nM, pyrrolidine dithiocarbamate (PDTC, 50 μM, TargetMol) was applied as an inhibitor of NF-κb (nuclear factor kappa), and C29 was used as a blocker of TLR2 signaling (50 μM, TargetMol).

Techniques: Migration, Transwell Assay, Blocking Assay

Journal: Journal of Neuroinflammation

Article Title: CXCL12 is involved in α-synuclein-triggered neuroinflammation of Parkinson’s disease

doi: 10.1186/s12974-019-1646-6

Figure Lengend Snippet: CXCL12/CXCR4 mediated the migration of microglia through FAK/Src/Rac-1 signaling. a The expression levels of GTP-Rac1 and total Rac1 were detected by western blotting after 24 h of stimulation by CXCL12 with or without AMD3100. b BV-2 cells were pretreated with NSC23766 for 2 h, and the migration of BV-2 cells towards CXCL12 was measured by the Transwell assay. c Western blot analysis was used to assess the expression levels of phospho-FAK (pY397), FAK, phospho-Src (pY416), and Src after 24 h of stimulation with CXCL12. d Migration of primary microglia towards CXCL12 with or without PP2 or PF573228 was measured by the Transwell assay. e Expression of GTP-Rac1 and total Rac1 were detected by western blotting after 24 h of stimulation by CXCL12 with or without PP2 or PF573228. The data are shown as the mean ± SEM from three independent experiments. **** p < 0.0001. *** p < 0.001. ** p < 0.01. * p < 0.05. BSA served as a control

Article Snippet: AMD3100 (1 ng/ml, TargetMol, USA) was used as a CXCR4 antagonist, TAK242 (Merck, Germany) was used as a TLR4 inhibitor at a concentration of 100 nM, pyrrolidine dithiocarbamate (PDTC, 50 μM, TargetMol) was applied as an inhibitor of NF-κb (nuclear factor kappa), and C29 was used as a blocker of TLR2 signaling (50 μM, TargetMol).

Techniques: Migration, Expressing, Western Blot, Transwell Assay, Control

Journal: Journal of Neuroinflammation

Article Title: CXCL12 is involved in α-synuclein-triggered neuroinflammation of Parkinson’s disease

doi: 10.1186/s12974-019-1646-6

Figure Lengend Snippet: Schematic of the mechanism by which α-synuclein induces the accumulation of microglia through CXCL12/CXCR4. Released α-syn establishes a gradient in the space between the neurons and the microglia. α-Syn aggregates may bind to the TLR4 on microglia, eliciting an overexcretion of CXCL12 through TLR4/IκB-α/NF-κB signaling. CXCL12 participates in α-synuclein-induced microglial migration through binding to CXCR4 through the CXCL12/CXCR4/FAK/Src/Rac1 pathway. As a result, the microglia continue to migrate along the concentration gradient of α-syn and CXCL12 toward the sources of α-syn

Article Snippet: AMD3100 (1 ng/ml, TargetMol, USA) was used as a CXCR4 antagonist, TAK242 (Merck, Germany) was used as a TLR4 inhibitor at a concentration of 100 nM, pyrrolidine dithiocarbamate (PDTC, 50 μM, TargetMol) was applied as an inhibitor of NF-κb (nuclear factor kappa), and C29 was used as a blocker of TLR2 signaling (50 μM, TargetMol).

Techniques: Migration, Binding Assay, Concentration Assay