Journal:

Article Title: Immortalized Mouse Inner Ear Cell Lines Demonstrate a Role for Chemokines in Promoting the Growth of Developing Statoacoustic Ganglion Neurons

doi: 10.1007/s10162-005-0013-8

Figure Lengend Snippet: Protein array analysis of (A) IMO2B1-conditioned media and (B) control medium. An array was section in order to test unconditioned medium and negative control IMO3A1 at the same time (B). Control medium that was not exposed to IMO cells (top, B) and IMO3A1-conditioned medium that failed to promote neurite outgrowth from chick SAG (batch 11.23; bottom, B) only detected the manufacturer's positive controls (biotinylated HRP conjugate, +) and low levels of gamma interferon (G). All other cytokines were below the level of detection in these media. In contrast, soluble TNF receptor 1 (T), gamma interferon (G), MCP-1 (M), and low levels of RANTES were detected in IMO2B1-conditioned medium shown to promote neurite outgrowth (A, batch 9.13). (− indicates negative controls, + positive controls).

Article Snippet: Lane 1: recombinant mouse MCP-1 (100 ng/ml; R&D Systems); lane 2: IMO2B1-conditioned medium of (batch 4.22); lane 3: IMO2B1-conditioned medium (batch 4.25); lane 4: IMO2B1 (batch 4.28); lane 5: IMO2B1-conditioned medium (batch 5.11); lane 6: recombinant mouse MCP-1 (200 ng/ml, R&D Systems); lane 7: IMO2B1-conditioned medium (batch 4.22 concentrated); lane 8: IMO2B1-conditioned medium (batch 4.25, concentrated); lane 9: IMO2B1-conditioned medium (batch 4.28 concentrated); lane 10: IMO2B1-conditioned medium (batch 5.11 concentrated).

Techniques: Protein Array, Negative Control

Journal:

Article Title: Immortalized Mouse Inner Ear Cell Lines Demonstrate a Role for Chemokines in Promoting the Growth of Developing Statoacoustic Ganglion Neurons

doi: 10.1007/s10162-005-0013-8

Figure Lengend Snippet: Immunofluorescence (green) representing the secondary antibody binding to the hair cell marker myosin VI was detected in hair cells of dissociated E14 mouse inner ear cultures. The dissociated cells form aggregates of hair cells and supporting cells. MCP-1 was also prominently expressed in hair cells (red), as well as in some unidentified cells surrounding the aggregate. Supporting cells within the aggregate were negative for MCP-1. The third panel demonstrates the areas where myosin VI and MCP-1 immunofluorescence overlaps (yellow). These results demonstrate that hair cells express MCP-1. Magnification, ×630.

Article Snippet: Lane 1: recombinant mouse MCP-1 (100 ng/ml; R&D Systems); lane 2: IMO2B1-conditioned medium of (batch 4.22); lane 3: IMO2B1-conditioned medium (batch 4.25); lane 4: IMO2B1 (batch 4.28); lane 5: IMO2B1-conditioned medium (batch 5.11); lane 6: recombinant mouse MCP-1 (200 ng/ml, R&D Systems); lane 7: IMO2B1-conditioned medium (batch 4.22 concentrated); lane 8: IMO2B1-conditioned medium (batch 4.25, concentrated); lane 9: IMO2B1-conditioned medium (batch 4.28 concentrated); lane 10: IMO2B1-conditioned medium (batch 5.11 concentrated).

Techniques: Immunofluorescence, Binding Assay, Marker

Journal:

Article Title: Immortalized Mouse Inner Ear Cell Lines Demonstrate a Role for Chemokines in Promoting the Growth of Developing Statoacoustic Ganglion Neurons

doi: 10.1007/s10162-005-0013-8

Figure Lengend Snippet: Western blot analysis demonstrated MCP-1-like protein in active IMO2B1-conditioned medium when concentrated by filtration column chromatography. Lane 1: recombinant mouse MCP-1 (100 ng/ml; R&D Systems); lane 2: IMO2B1-conditioned medium of (batch 4.22); lane 3: IMO2B1-conditioned medium (batch 4.25); lane 4: IMO2B1 (batch 4.28); lane 5: IMO2B1-conditioned medium (batch 5.11); lane 6: recombinant mouse MCP-1 (200 ng/ml, R&D Systems); lane 7: IMO2B1-conditioned medium (batch 4.22 concentrated); lane 8: IMO2B1-conditioned medium (batch 4.25, concentrated); lane 9: IMO2B1-conditioned medium (batch 4.28 concentrated); lane 10: IMO2B1-conditioned medium (batch 5.11 concentrated). Molecular weight standards (kDa) are indicated at the left.

Article Snippet: Lane 1: recombinant mouse MCP-1 (100 ng/ml; R&D Systems); lane 2: IMO2B1-conditioned medium of (batch 4.22); lane 3: IMO2B1-conditioned medium (batch 4.25); lane 4: IMO2B1 (batch 4.28); lane 5: IMO2B1-conditioned medium (batch 5.11); lane 6: recombinant mouse MCP-1 (200 ng/ml, R&D Systems); lane 7: IMO2B1-conditioned medium (batch 4.22 concentrated); lane 8: IMO2B1-conditioned medium (batch 4.25, concentrated); lane 9: IMO2B1-conditioned medium (batch 4.28 concentrated); lane 10: IMO2B1-conditioned medium (batch 5.11 concentrated).

Techniques: Western Blot, Filtration, Column Chromatography, Recombinant, Molecular Weight

Journal:

Article Title: Immortalized Mouse Inner Ear Cell Lines Demonstrate a Role for Chemokines in Promoting the Growth of Developing Statoacoustic Ganglion Neurons

doi: 10.1007/s10162-005-0013-8

Figure Lengend Snippet: E5 chick SAG treated with IMO2B1 produced neurite outgrowth (mean 2.6, SE 0.6, n = 5,batch 9.22). Addition of 150 ng/ml of anti-MCP-1 antibody to the IMO2B1 medium decreased outgrowth of SAG significantly (mean 0.6, SE 0.3, n = 5, p < 0.01, batch 9.22).

Article Snippet: Lane 1: recombinant mouse MCP-1 (100 ng/ml; R&D Systems); lane 2: IMO2B1-conditioned medium of (batch 4.22); lane 3: IMO2B1-conditioned medium (batch 4.25); lane 4: IMO2B1 (batch 4.28); lane 5: IMO2B1-conditioned medium (batch 5.11); lane 6: recombinant mouse MCP-1 (200 ng/ml, R&D Systems); lane 7: IMO2B1-conditioned medium (batch 4.22 concentrated); lane 8: IMO2B1-conditioned medium (batch 4.25, concentrated); lane 9: IMO2B1-conditioned medium (batch 4.28 concentrated); lane 10: IMO2B1-conditioned medium (batch 5.11 concentrated).

Techniques: Produced

Journal:

Article Title: Immortalized Mouse Inner Ear Cell Lines Demonstrate a Role for Chemokines in Promoting the Growth of Developing Statoacoustic Ganglion Neurons

doi: 10.1007/s10162-005-0013-8

Figure Lengend Snippet: (A) IMO2B1-conditioned medium promoted neurite outgrowth of E5 chick SAG (batch 9.22; explant score = 4). (B) Addition of anti-MCP-1 antibody (150 ng/ml) decreased E5 chick SAG outgrowth (explant score = 1.5). The photo shows the area of maximal outgrowth. Magnification, ×100 (A), ×200 (B).

Article Snippet: Lane 1: recombinant mouse MCP-1 (100 ng/ml; R&D Systems); lane 2: IMO2B1-conditioned medium of (batch 4.22); lane 3: IMO2B1-conditioned medium (batch 4.25); lane 4: IMO2B1 (batch 4.28); lane 5: IMO2B1-conditioned medium (batch 5.11); lane 6: recombinant mouse MCP-1 (200 ng/ml, R&D Systems); lane 7: IMO2B1-conditioned medium (batch 4.22 concentrated); lane 8: IMO2B1-conditioned medium (batch 4.25, concentrated); lane 9: IMO2B1-conditioned medium (batch 4.28 concentrated); lane 10: IMO2B1-conditioned medium (batch 5.11 concentrated).

Techniques:

Journal:

Article Title: Immortalized Mouse Inner Ear Cell Lines Demonstrate a Role for Chemokines in Promoting the Growth of Developing Statoacoustic Ganglion Neurons

doi: 10.1007/s10162-005-0013-8

Figure Lengend Snippet: Monocyte migration was measured using a classic chemotactic assay. The number of migrating cells is depicted minus the background number of cells migrating in control conditions (mean 58 cells, SE 19, n = 7). In the presence of MCP-1 (20 ng/ml), monocytes migrated to the side of the chamber containing the protein (mean 299, SE, 40, n = 4). Addition of the function-blocking anti-MCP-1 antibody reduced the migration of the monocytes significantly (mean 46, SE 31, n = 3, p < 0.01). IMO2B1 alone also induced the migration of monocytes above background levels (mean 58, SE 14, n = 7, batch 2.05) and the function-blocking antibody decreased this migration significantly (mean 11, SE 8, n = 6, p < 0.01).

Article Snippet: Lane 1: recombinant mouse MCP-1 (100 ng/ml; R&D Systems); lane 2: IMO2B1-conditioned medium of (batch 4.22); lane 3: IMO2B1-conditioned medium (batch 4.25); lane 4: IMO2B1 (batch 4.28); lane 5: IMO2B1-conditioned medium (batch 5.11); lane 6: recombinant mouse MCP-1 (200 ng/ml, R&D Systems); lane 7: IMO2B1-conditioned medium (batch 4.22 concentrated); lane 8: IMO2B1-conditioned medium (batch 4.25, concentrated); lane 9: IMO2B1-conditioned medium (batch 4.28 concentrated); lane 10: IMO2B1-conditioned medium (batch 5.11 concentrated).

Techniques: Migration, Chemotaxis Assay, Blocking Assay

Journal:

Article Title: Immortalized Mouse Inner Ear Cell Lines Demonstrate a Role for Chemokines in Promoting the Growth of Developing Statoacoustic Ganglion Neurons

doi: 10.1007/s10162-005-0013-8

Figure Lengend Snippet: IMO2B1-conditioned medium produced outgrowth from E5 chick SAG (IMO2B1 batch 9.13, mean 3.4, SE 0.35, n = 7). When anti-MCP-1 function-blocking antibody was added at a concentration of 10 or 25 ng/ml (n = 14 total) the amount of outgrowth was reduced significantly (mean 1.97, SE 0.42, n = 14, p < 0.01). Supplementing the anti-MCP-1-treated IMO2B1-conditioned medium with MCP-1 (“add back,” 20 ng/ml) led to a statistically significant increase in SAG neurite outgrowth (mean 3.72, SE 0.31, n = 11, p < 0.01) above that observed with anti-MCP-1 only, indicating a direct role for MCP-1 in promoting SAG outgrowth. No further increase was noted when 200 ng/ml of MCP-1 (mean 2.7, SE 0.42, n = 5; p > 0.01) was added to the antibody-treated cultures.

Article Snippet: Lane 1: recombinant mouse MCP-1 (100 ng/ml; R&D Systems); lane 2: IMO2B1-conditioned medium of (batch 4.22); lane 3: IMO2B1-conditioned medium (batch 4.25); lane 4: IMO2B1 (batch 4.28); lane 5: IMO2B1-conditioned medium (batch 5.11); lane 6: recombinant mouse MCP-1 (200 ng/ml, R&D Systems); lane 7: IMO2B1-conditioned medium (batch 4.22 concentrated); lane 8: IMO2B1-conditioned medium (batch 4.25, concentrated); lane 9: IMO2B1-conditioned medium (batch 4.28 concentrated); lane 10: IMO2B1-conditioned medium (batch 5.11 concentrated).

Techniques: Produced, Blocking Assay, Concentration Assay

Journal: Frontiers in Immunology

Article Title: CCR2 + Macrophages Promote Orthodontic Tooth Movement and Alveolar Bone Remodeling

doi: 10.3389/fimmu.2022.835986

Figure Lengend Snippet: CCR2/CCL2 axis promoted pro-inflammatory macrophages through the phosphorylation of p65. (A) Relative mRNA expression of Nos2 in untreated, CCL2 treated and LPS treated macrophages. Data is from 3 independent experiments. Values are mean ± SD. *P < 0.05. **P <0.01. (B) Representative immunofluorescence staining of iNOS in untreated, CCL2 treated and LPS treated macrophages. (C) Relative mRNA expression of inflammatory factors in macrophages treated with LPS or IL-4, and RS504393. Data is from 3 independent experiments. Values are mean ± SD. *P < 0.05. ns, no significance. (D) Relative mRNA expression of Ccr2 in macrophages treated with CCL2 or not, and with Ccr2 siRNA. Data is from 3 independent experiments. Values are mean ± SD. *P < 0.05. (E, F) Relative mRNA expression of pro-inflammatory (E) and anti-inflammatory factors (F) in macrophages treated with CCL2 or not, and with Ccr2 siRNA. Data is from 3 independent experiments. Values are mean ± SD. *P < 0.05. ns, no significance. (G) Representative immunofluorescence staining of p65 in macrophages in untreated, treated with CCL2, and CCL2+RS504393. Data is from 3 independent experiments. Values are mean ± SD. *P < 0.05. **P <0.01. (H) Relative mRNA expression of inflammatory factors Il6 , Il1β and nos2 treated with CCL2 or p65 inhibitor. Data is from 3 independent experiments. Values are mean ± SD. *P < 0.05, ns, no significance.

Article Snippet: BMDMs were pretreated with P65 inhibitor Helenalin (10μm, HY-119970; MCE) for 2 hours, then treated with CCL2 (100ng/ml, 479-JE; R&D) for 3 hours.

Techniques: Expressing, Immunofluorescence, Staining

Journal: Frontiers in Immunology

Article Title: CCR2 + Macrophages Promote Orthodontic Tooth Movement and Alveolar Bone Remodeling

doi: 10.3389/fimmu.2022.835986

Figure Lengend Snippet: CCL2 treatment promoted the phosphorylation of p65 in macrophages. (A) Western blot analysis of p-p65 and p65 expression in macrophages treated with CCL2 for 0, 30, 60 and 360 minutes. Data is from 3 independent experiments. Densitometric quantitation with Image J. Values are mean ± SD. *P < 0.05. (B) Western blot analysis of p-p65 and p65 expression in macrophages treated with CCL2 and RS504393. Data is from 3 independent experiments. Densitometric quantitation with Image J. Values are mean ± SD. *P < 0.05. ns, no significance. (C, D) Western bolt analysis of p-p65 and p65 expression in macrophages treated with CCL2 or not, and with Ccr2 siRNA. Data is from 3 independent experiments. Densitometric quantitation with Image J. Values are mean ± SD. *P < 0.05. (E, F) The distance of OTM in TM and TM+CCR2i group and representative 3D micro-CT reconstruction. White arrow: direction of force and tooth movement. Values are mean ± SD. n = 5. ***P < 0.001. (G) Graphic abstract of this study.

Article Snippet: BMDMs were pretreated with P65 inhibitor Helenalin (10μm, HY-119970; MCE) for 2 hours, then treated with CCL2 (100ng/ml, 479-JE; R&D) for 3 hours.

Techniques: Western Blot, Expressing, Quantitation Assay, Micro-CT

Journal: Experimental & Molecular Medicine

Article Title: CCL12 induces trabecular bone loss by stimulating RANKL production in BMSCs during acute lung injury

doi: 10.1038/s12276-023-00970-w

Figure Lengend Snippet: a Chemokine levels in the peripheral blood of ALI mice on Days 0, 2, 8, and 14 after LPS administration were determined by an antibody array ( n = 6). b Schematic diagram of mice treated with 5 mg/kg LPS (i.t.) and 4 mg/kg neutralizing antibodies against CCL12 via the tail vein ( n = 8). Saline was used as a negative control for LPS. Isotype IgG was used as a negative control for the neutralizing antibody. The day when LPS was administered was defined as “Day 0”. A neutralizing antibody against CCL12 was injected for the first time before LPS administration on Day 0. Then, the antibody was injected every 4 days. Femurs were collected on Day 14. c Representative 3D reconstruction images of the distal femur trabecular bone of ALI mice after the administration of the neutralizing antibody against CCL12 were determined by micro-CT. d BV/TV, Tb.N, and Tb.Sp of the distal femur in ALI mice after the administration of the neutralizing antibody against CCL12 ( n = 8). e H&E staining and histomorphometric analysis of the Ob.N/B.Pm and Ob.S/BS in the femurs of ALI mice after the administration of the neutralizing antibody against CCL12 ( n = 8). Scale bar: 40 μm. f TRAP staining and histomorphometric analysis of the Oc.N/B.Pm and Oc.S/BS in the femurs of ALI mice after the administration of the neutralizing antibody against CCL12 ( n = 8). Scale bar: 40 μm. The data are representative of three independent experiments. The data are shown as the means ± s.d. * p < 0.05, ** p < 0.01, *** p < 0.001, ns no significance.

Article Snippet: BMSCs were treated in vitro with 50 and 100 ng/ml recombinant mouse CCL12 (R&D Systems, Minneapolis, MN).

Techniques: Ab Array, Saline, Negative Control, Injection, Micro-CT, Staining

Journal: Experimental & Molecular Medicine

Article Title: CCL12 induces trabecular bone loss by stimulating RANKL production in BMSCs during acute lung injury

doi: 10.1038/s12276-023-00970-w

Figure Lengend Snippet: a CCL12 levels in the peripheral blood of wild-type (WT) and CCL12 −/− mice were determined by ELISA ( n = 8). b , c CCL12 protein expression in the bone marrow of WT and CCL12 −/− mice was determined by immunohistochemistry ( b ) and ELISA ( c ) ( n = 8). B: bone. Scale bar: 20 μm. d Representative 3D reconstruction images of the distal femur trabecular bones of WT and CCL12 −/− mice were determined by micro-CT. e BV/TV, Tb.N, and Tb.Sp of the distal femurs of WT and CCL12 −/− mice ( n = 8). f H&E staining and histomorphometric analysis of the Ob.N/B.Pm and Ob.S/BS in the femur ( n = 8). Scale bar: 40 μm. g TRAP staining and histomorphometric analysis of the Oc.N/B.Pm and Oc.S/BS in the femurs of WT and CCL12 −/− mice (n = 8). Scale bar: 40 μm. The data are representative of three independent experiments. The data are shown as the means ± s.d. * p < 0.05, ** p < 0.01, *** p < 0.001, ns no significance.

Article Snippet: BMSCs were treated in vitro with 50 and 100 ng/ml recombinant mouse CCL12 (R&D Systems, Minneapolis, MN).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Immunohistochemistry, Micro-CT, Staining

Journal: Experimental & Molecular Medicine

Article Title: CCL12 induces trabecular bone loss by stimulating RANKL production in BMSCs during acute lung injury

doi: 10.1038/s12276-023-00970-w

Figure Lengend Snippet: a RANKL levels in peripheral blood and bone marrow of ALI mice were determined by ELISA ( n = 8). b RANKL protein expression in the femur osteocytes, osteoblasts, and bone marrow of ALI mice was determined by immunohistochemistry ( n = 8). B: bone. Scale bar: 20 μm. c OPG levels in the peripheral blood and bone marrow of ALI mice were determined by ELISA ( n = 8). d OPG protein expression in the femur osteocytes, osteoblasts, and bone marrow of ALI mice was determined by immunohistochemistry ( n = 8). B: bone. Scale bar: 20 μm. e RANKL levels in the peripheral blood and bone marrow of ALI mice with global deletion of CCL12 were determined by ELISA ( n = 8). f RANKL protein expression in the femur osteocytes, osteoblasts, and bone marrow of ALI mice with global deletion of CCL12 was determined by immunohistochemistry ( n = 8). B: bone. Scale bar: 20 μm. The data are representative of three independent experiments. The data are shown as the means ± s.d. ** p < 0.01, *** p < 0.001, ns no significance.

Article Snippet: BMSCs were treated in vitro with 50 and 100 ng/ml recombinant mouse CCL12 (R&D Systems, Minneapolis, MN).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Immunohistochemistry

Journal: Experimental & Molecular Medicine

Article Title: CCL12 induces trabecular bone loss by stimulating RANKL production in BMSCs during acute lung injury

doi: 10.1038/s12276-023-00970-w

Figure Lengend Snippet: a RANKL mRNA and protein expression in RANKL + CCR2 + BMSCs from ALI mice ( n = 8). b RANKL mRNA and protein expression in primary BMSCs and ST2 cells cultured with 0, 50, and 100 ng/ml recombinant CCL12 for 48 h in vitro ( n = 8). c RANKL mRNA and protein expression in RANKL + CCR2 + BMSCs from ALI mice with global deletion of CCL12 ( n = 8). d RANKL protein expression in the femur osteocytes, osteoblasts, and bone marrow of ALI mice with conditional deletion of CCR2 in BMSCs was determined by immunohistochemistry ( n = 8). B: bone. Scale bar: 20 μm. e RANKL mRNA and protein expression in BMSCs with CCR2 deletion from ALI mice was determined by flow cytometry ( n = 8). f TRAP staining and histomorphometric analysis of the Oc.N/B.Pm and Oc.S/BS in the femurs of ALI mice with conditional deletion of CCR2 in BMSCs ( n = 8). Scale bar: 40 μm. g Representative 3D reconstruction images and the BV/TV, Tb.N, and Tb.Sp of the distal femur trabecular bones of ALI mice with conditional deletion of CCR2 in BMSCs were determined by micro-CT ( n = 8). α-Tubulin was used as an internal control for qPCR. The data are representative of three independent experiments. The data are shown as the means ± s.d. * p < 0.05, ** p < 0.01, *** p < 0.001, ns no significance.

Article Snippet: BMSCs were treated in vitro with 50 and 100 ng/ml recombinant mouse CCL12 (R&D Systems, Minneapolis, MN).

Techniques: Expressing, Cell Culture, Recombinant, In Vitro, Immunohistochemistry, Flow Cytometry, Staining, Micro-CT, Control

Journal: Experimental & Molecular Medicine

Article Title: CCL12 induces trabecular bone loss by stimulating RANKL production in BMSCs during acute lung injury

doi: 10.1038/s12276-023-00970-w

Figure Lengend Snippet: a Diagram of the mouse RANKL promoter. Analysis of transcription factor binding sites in the RANKL promoter revealed putative STAT3 and STAT4 binding sites around the transcription start site. b STAT3 binding to the ChIP-1 and ChIP-2 regions within the RANKL promoter in BMSCs in response to 0, 50, and 100 ng/ml recombinant CCL12 treatment for 24 h, as determined by ChIP. c STAT4 binding to the ChIP-1 and ChIP-2 regions within the RANKL promoter in BMSCs in response to 0, 50, and 100 ng/ml recombinant CCL12 treatment for 24 h, as determined by ChIP. d Lentivirus-mediated stable overexpression (OE) of STAT3 with an HA tag and STAT4 with a Flag tag in BMSC cultures was determined by western blotting. e STAT3 binding to the ChIP-1 region within the RANKL promoter in BMSCs with STAT3 overexpression was determined by ChIP. f STAT4 binding to the ChIP-2 region within the RANKL promoter in BMSCs with STAT4 overexpression was determined by ChIP. g Lentivirus-mediated stable knockdown of STAT3 and STAT4 in BMSC cultures was determined by western blotting. h STAT3 binding to the ChIP-1 region within the RANKL promoter in BMSCs treated with recombinant CCL12 for 24 h and/or STAT3 knockdown was determined by ChIP. i STAT4 binding to the ChIP-2 region within the RANKL promoter in BMSCs treated with recombinant CCL12 for 24 h and/or STAT4 knockdown was determined by ChIP. Isotype IgG was used as a negative control in the ChIP assay. α-Tubulin was used as a loading control for western blotting. The data are representative of three independent experiments. The data are shown as the means ± s.d. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: BMSCs were treated in vitro with 50 and 100 ng/ml recombinant mouse CCL12 (R&D Systems, Minneapolis, MN).

Techniques: Binding Assay, Recombinant, Over Expression, FLAG-tag, Western Blot, Knockdown, Negative Control, Control

Journal: Experimental & Molecular Medicine

Article Title: CCL12 induces trabecular bone loss by stimulating RANKL production in BMSCs during acute lung injury

doi: 10.1038/s12276-023-00970-w

Figure Lengend Snippet: a Phosphorylated Jak2 protein levels in BMSCs in response to 0, 50, and 100 ng/ml recombinant CCL12 treatment for 6 h were determined by western blotting. b Phosphorylated STAT3 protein levels in BMSCs in response to 0, 50, and 100 ng/ml recombinant CCL12 treatment for 6 h were determined by western blotting. c Phosphorylated STAT4 protein levels in BMSCs in response to 0, 50, and 100 ng/ml recombinant CCL12 treatment for 6 h were determined by western blotting. d Phosphorylated STAT3 protein levels in the nuclear fraction of BMSCs in response to 0, 50, and 100 ng/ml recombinant CCL12 treatment for 6 h were determined by western blotting. e Phosphorylated STAT4 protein levels in the nuclear fraction of BMSCs in response to 0, 50, and 100 ng/ml recombinant CCL12 treatment for 6 h were determined by western blotting. f RANKL mRNA expression in BMSCs in response to recombinant CCL12 (100 ng/ml) and/or AG490 (50 μM) treatment for 48 h ( n = 8). g RANKL mRNA expression in BMSCs in response to recombinant CCL12 (100 ng/ml) treatment for 48 h and/or STAT3/4 knockdown ( n = 8). h Luciferase activity of RANKL promoter deletion mutant-driven luciferase reporter gene vectors, including Luc (empty vector as negative control), −1005 bp/+156 bp (containing putative STAT3 and STAT4 binding sites), −454 bp/+156 bp (containing putative STAT4 binding site), and −1005 bp/−429 bp (containing putative STAT3 binding site), in BMSCs treated with recombinant CCL12 for 48 h ( n = 8). i Luciferase activity of RANKL promoter deletion mutant-driven luciferase reporter gene vectors, including Luc, STAT3 + STAT4 binding site, 3×STAT3 + STAT4 binding site, and STAT3 + 3×STAT4 binding site, in BMSCs treated with recombinant CCL12 for 48 h ( n = 8). j Luciferase activity of RANKL promoter deletion mutant-driven luciferase reporter gene vectors with and without pcDNA3.1-STAT3/4 vector cotransfection in BMSCs treated with recombinant CCL12 for 48 h ( n = 8). α-Tubulin was used as an internal control for qPCR and western blot analysis of total proteins. Lamin B1 was used as a loading control for western blot analysis of nuclear proteins. The Renilla luciferase vector was used as a control for transfection efficiency. The data are representative of three independent experiments. The data are shown as the means ± s.d. * p < 0.05, ** p < 0.01, *** p < 0.001, ns no significance.

Article Snippet: BMSCs were treated in vitro with 50 and 100 ng/ml recombinant mouse CCL12 (R&D Systems, Minneapolis, MN).

Techniques: Recombinant, Western Blot, Expressing, Knockdown, Luciferase, Activity Assay, Mutagenesis, Plasmid Preparation, Negative Control, Binding Assay, Cotransfection, Control, Transfection

Journal: Experimental & Molecular Medicine

Article Title: CCL12 induces trabecular bone loss by stimulating RANKL production in BMSCs during acute lung injury

doi: 10.1038/s12276-023-00970-w

Figure Lengend Snippet: LPS induces acute lung injury, and CCL12 levels are significantly elevated in the peripheral blood ( a ) and bone marrow ( b ) in response to inflammation. In bone marrow, CCL12 binds to CCR2 on the cell membrane of BMSCs to activate RANKL transcription through the Jak2/STAT4 axis ( b ). RANKL promotes osteoclastic differentiation in bone marrow monocytes ( c ), which results in enhanced bone resorption activity and trabecular bone loss in acute lung injury.

Article Snippet: BMSCs were treated in vitro with 50 and 100 ng/ml recombinant mouse CCL12 (R&D Systems, Minneapolis, MN).

Techniques: Membrane, Activity Assay

Journal: Cancer letters

Article Title: Induction of the MCP Chemokine Cluster Cascade in the Periphery by Cancer Cell-Derived CCL3

doi: 10.1016/j.canlet.2016.12.028

Figure Lengend Snippet: (A) Representative images of cytokine arrays of EO771 and 3T3 cell extracts and conditioned media. Arrows indicate cytokines upregulated in EO771 as compared to 3T3, >2 fold for media and >1 fold for cells. (B) Expression ratio of cytokines with >1.5 fold induction in the media. Data for MMP-3 and osteopontin are not shown. (C) Ccl8 levels in the supernatants of 3T3 cells exposed to the factors indicated at concentration 10 ng/ml for 24 hours. Results are expressed as average + SEM. *, p<0.05.

Article Snippet: Cell supernatants were collected 24 hours later and Ccl8 protein levels were assessed by Mouse CCL8/MCP-2 DuoSet ELISA Development kit (R&D Systems) according to manufacturer’s protocol.

Techniques: Expressing, Concentration Assay

Journal: Neuro-oncology Advances

Article Title: Immune deconvolution and temporal mapping identifies stromal targets and developmental intervals for abrogating murine low-grade optic glioma formation

doi: 10.1093/noajnl/vdab194

Figure Lengend Snippet: CD8 + T cell predominance is governed by NF1/RAS regulation of Ccr4 expression. A, CD8 + T cells content in Nf1 -OPG optic nerves ( n = 10) is similar to controls ( n = 8) at 3, 4.5, 6, and 9 WOA, but increases by 12 WOA ( P < 0.0001). Nf1 +/– mice ( n = 6) have similar numbers of CD8 + T cells as controls from 3 to 12 WOA. B, CD4 + T cell content does not change in Nf1 -OPG optic nerves relative to controls at 3, 4.5, 6, 9, and 12 WOA. C, Ccl2 did not increase CD4 + T cell migration in WT or Nf1 +/– mice. Ccl12 or medium (control) does not increase CD4 + T cell migration. Ccl2 increased Nf1 +/– CD8 + T cell migration relative to WT controls ( P < 0.0001). Nf1 +/- and WT CD8 + T cells treated with medium (Control) or Ccl12 exhibit similar migration. D, Nf1 +/- and WT CD4 + T cells had similar levels of Ccr4 expression. Nf1 +/– CD8 + T cells have increased Ccr4 expression relative to their WT counterparts ( P = 0.0474). E, Ccr4 inhibition (AZD2098; 15µM) in CD8 + T cells decreases Ccl2-induced, but not Ccl12-induced, migration ( P = 0.0063). F, Nf1 +/– and WT CD4 + T cells had similar levels of Ras activity. Nf1 +/– CD8 + T cells have increased Ras activity relative to their WT counterparts ( P = 0.0363) G, Inhibition of Ras activity (10µM IN-1) reduces Ccl2-induced,but not Ccl12-induced, CD8 + T cell migration ( P = 0.0104). H, RAS activity inhibition (10µM IN-1) decreases Ccr4 expression in Nf1+/- CD8 + T cells ( P = 0.0069). I, Proposed model of Ccl2 signaling through Ccr4 in Nf1+/– CD8 + T cells.

Article Snippet: 500 μl of chemoattractant media containing Ccl2 (20ng/ml, 479-JE-050/CF-R&D systems) or Ccl12 (25ng/ml, 428-P5-025-R&D systems) was added to the lower chamber , and the number of T cells in the lower chambers counted by microscopy at 20x after 6 h.

Techniques: Expressing, Migration, Control, Inhibition, Activity Assay