Journal: Molecular human reproduction

Article Title: IL-33 enhances proliferation and invasiveness of decidual stromal cells by up-regulation of CCL2/CCR2 via NF-κB and ERK1/2 signaling.

doi: 10.1093/molehr/gat094

Figure Lengend Snippet: Figure 4 Effects of IL-33/ST2L/sST2 axis on CCL2/CCR2 expres- sion in DSCs. The secretion of chemokine CCL2 (A) by DSCs was determined by ELISA, and the expression of CCR2 on DSCs (B) by flow cytometry. DSCs (2 × 105 cells/well) were seeded in 24-well plates with or without rhIL-33(0.01–10 ng/ml) and sST2 (100– 400 ng/ml). The supernatant was then collected after 48 h while DSCs were used to detect the protein concentration, and the CCL2 levelofeachgroupwascalculatedastheratiooftheCCL2concentration of supernatant to the protein concentration. The pictures are from a representative experiment and the numbers are the percentages of positive cells. Results are representative of three independent experi- ments. ***P , 0.001. CCL2-APC, allophycocyanin labeled CCL2 antibody.

Article Snippet: BrdU cell proliferation assay Bromodeoxyuridine (5-bromo-2′-deoxyuridine, BrdU) cell proliferation assay was applied to evaluate the effect of IL-33 on cell proliferation with or without sST2, anti-CCL2 neutralizing antibody (MAB279, R&D Systems) and CCR2 blocker RS102895 (R1903, Sigma-Aldrich, Santa Clara, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Cytometry, Protein Concentration, Labeling

Journal: Molecular human reproduction

Article Title: IL-33 enhances proliferation and invasiveness of decidual stromal cells by up-regulation of CCL2/CCR2 via NF-κB and ERK1/2 signaling.

doi: 10.1093/molehr/gat094

Figure Lengend Snippet: Figure 5 The increased proliferation and invasiveness of DSCs induced by IL-33 are dependent on CCL2/CCR2 interaction. DSCs (1 × 104 cell/well, BrdUproliferationassay;3 × 105 cells/well,cellcycleassay;2 × 105 cells/well,invasionassay)weretreatedwithrhIL-33(1 ng/ml),a-CCL2(1 mg/ml),or CCR2 blocker RS102895(100 ng/ml) with vehicle as control. After stimulation with rhIL-33 (1 ng/ml), the cells were treated with CCL2 neutralizing anti- body (1 mg/ml) or RS102895 (100 ng/ml) foranother 48 h. Thereafter, BrdU proliferation assay(A), cell cycle (B) and invasion assay (C) wereconducted to analyze proliferation and invasiveness of DSCs, respectively. Error bars depict the standard deviation of the sample mean. The images are from an ex- periment that is representative of three independently conducted experiments. The x-axis, from the left, represents the cell cycle phases of G1(green region), S(the region between G1 and G2) and G2(blue region). *P , 0.05, **P , 0.01, ***P , 0.001.

Article Snippet: BrdU cell proliferation assay Bromodeoxyuridine (5-bromo-2′-deoxyuridine, BrdU) cell proliferation assay was applied to evaluate the effect of IL-33 on cell proliferation with or without sST2, anti-CCL2 neutralizing antibody (MAB279, R&D Systems) and CCR2 blocker RS102895 (R1903, Sigma-Aldrich, Santa Clara, USA).

Techniques: Control, Proliferation Assay, Invasion Assay, Standard Deviation

Journal: Molecular human reproduction

Article Title: IL-33 enhances proliferation and invasiveness of decidual stromal cells by up-regulation of CCL2/CCR2 via NF-κB and ERK1/2 signaling.

doi: 10.1093/molehr/gat094

Figure Lengend Snippet: Figure 8 Schematic representation of IL-33 effects on regulating biological behavior of DSCs. IL-33 can stimulate CCL2/CCR2 expression through NF-kB and ERK1/2 signal pathways, thereby enhancing proliferation and invasion of DSCs. At the same time, proliferation relative gene (PCNA, survivin) and invasion relative gene (titin, MMP2) are also increased in response to IL-33, which further promotes prolifertation and invasiveness of DSCs. sST2 can block IL-33/ST2 signaling through bounding to IL-33.

Article Snippet: BrdU cell proliferation assay Bromodeoxyuridine (5-bromo-2′-deoxyuridine, BrdU) cell proliferation assay was applied to evaluate the effect of IL-33 on cell proliferation with or without sST2, anti-CCL2 neutralizing antibody (MAB279, R&D Systems) and CCR2 blocker RS102895 (R1903, Sigma-Aldrich, Santa Clara, USA).

Techniques: Expressing, Blocking Assay

Journal: Frontiers in Pharmacology

Article Title: Ningmitai capsules have anti-inflammatory and pain-relieving effects in the chronic prostatitis/chronic pelvic pain syndrome mouse model through systemic immunity

doi: 10.3389/fphar.2022.949316

Figure Lengend Snippet: Antibody information used in the article.

Article Snippet: MCP1/CCL2 (5H2) Mouse mAb , 1:1,000(WB) , ZENBIO(220691).

Techniques:

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: Impact of prolonged stimulation by Tumor CM on the release of inflammatory chemokines by the resulting CAFs. Human BM-derived MSCs were cultured with Tumor CM from MDA-MB-231 cells (MDA) (A) or MCF-7 cells (B) over a prolonged period of time (~30 days; MSCs + MDA CM or MSCs + MCF-7 CM, respectively). Twenty-four hours after medium exchange to fresh Tumor CM, cell supernatants were collected and the expression of CCL2 (A1, B1), CXCL8 (A2, B2) and CCL5 (A3, B3) was determined in comparison with supernatants of MSCs that were not supplemented with CM (MSCs) and with the original Tumor CM of MDA-MB-231 or MCF-7 cells alone (MDA CM or MCF-7 CM, respectively). Chemokine expression was determined by ELISA, in the linear range of absorbance. (A1), (A2), (B1) Representatives of n = 3 independent experiments that have shown similar results. (A3), (B2), (B3) Ratios between MSCs and MSCs + Tumor CM were not consistent in different experimental repeats. Therefore, in these panels, the findings are presented as mean ± standard deviation of normalized values (MSCs were given the value of 1) obtained in relevant experimental repeats (at least n = 3).

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Derivative Assay, Cell Culture, Expressing, Comparison, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: TNF-α induces potent elevation in inflammatory traits in MSCs and Tumor-CM-generated CAFs. Human BM-derived MSCs were cultured with Tumor CM from MDA-MB-231 cells (MDA) (A) or from MCF-7 cells (B) over a prolonged period of time (~30 days). TNF-α (50 ng/ml) or its vehicle (in control cells) was added for the last 24 hours. Expression of CCL2, CXCL8 and CCL5 was then determined in supernatants of the cells. (A) Expression of the chemokines in the four experimental groups included in the study: MSCs grown in culture for ~30 days without any additional stimulus (MSCs); MSCs grown in culture for ~30 days in the presence of Tumor CM derived from MDA-MB-231 cells (MSCs + MDA CM); MSCs grown in culture for ~30 days and stimulated by TNF-α at the last 24 hours of culture (MSCs + TNF-α); and MSCs grown in culture for ~30 days in the presence of Tumor CM derived from MDA-MB-231 cells and stimulated by TNF-α at the last 24 hours of culture (MSCs + MDA CM + TNF-α). Expression of CCL2 (A1), CXCL8 (A2) and CCL5 (A3) was determined by ELISA, in the linear range of absorbance. (B) Experimental design as in (A), but with MCF-7-derived CM. In each panel, the findings are representatives of at least n = 3 experiments that have shown similar results. In comparisons between MSCs and all other groups: * P <0.05, ** P ≤0.01, *** P ≤0.001. NS, not significant. # Differences between the two indicated groups have shown variability in n ≥ 3 independent experiments and overall were not significant.

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Generated, Derivative Assay, Cell Culture, Control, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: Basal and cytokine-induced release of inflammatory chemokines by patient CAFs. CAFs were isolated from lung metastasis of a breast cancer patient (CAFs #1) or from a primary tumor of a different breast cancer patient (CAFs #2). (A1, B1) Expression of the pro-malignancy chemokines CCL2, CXCL8 and CCL5 was determined by ELISA, in the linear range of absorbance. (A2, B2) Expression of CCL5 was determined following TNFa and IL-1β stimulation (TNF-α, 50 ng/ml; IL-1β, 500 pg/ml; 48 hours). Control cells were stimulated by vehicle. Expression of the chemokines was determined by ELISA, in the linear range of absorbance. In all panels, the findings are representatives of at least n = 3 independent experiments that have shown similar results.

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: TNF-α upregulates the inflammatory profile of MSCs via receptors TNF-RI and TNF-RII. (A) Relative induction of CCL2, CXCL8 and CCL5 in MSCs by TNF-α. Human BM-derived MSCs were stimulated by TNF-α (50 ng/ml) for 24 hours and the expression of the inflammatory chemokines CCL2, CXCL8 and CCL5 in supernatants of MSCs was determined by ELISA, in the linear range of absorbance. Results are from an experiment in which all three chemokines were analyzed in parallel, and the ratios between the three chemokines are representatives of the values obtained in many experimental repeats. (B) Human BM-derived MSCs were exposed to neutralizing antibodies against tumor necrosis factor receptors TNF-RI and TNF-RII or nonrelevant isotype-matched control antibodies (Control Ab) 1 hour prior to stimulation with TNF-α (50 ng/ml), and in the course of 24 hours of stimulation by the cytokine. Control, cells stimulated with vehicle. Expression of CCL2 was determined by ELISA, in the linear range of absorbance. The findings are representatives of at least n = 3 independent experiments that have shown similar results.

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Derivative Assay, Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: CCL2 is expressed by CAFs localized in vicinity to breast cancer cells in tumors of invasive ductal carcinoma (IDC) patients. CCL2 expression was determined by immunohistochemistry in biopsy sections of patients diagnosed with IDC: (A) IDC #1, (B) IDC #2. CCL2 staining was detected in cancer cells and in adjacent fibroblasts. Some of the CCL2-expressing CAFs are indicated by arrows.

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: Induction of CCL2 and CLXL8 in TNFα-stimulated MSCs is not mediated via the AP-1 pathway. (A) Human BM-derived MSCs were stimulated by TNF-α (50 ng/ml) for 5 and 10 minutes. Control cells were treated by the vehicle of TNF-α. c-Jun levels and phosphorylation were determined by western blot (WB) analyses. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. (B) Human BM-derived MSCs were transiently transfected by small interfering RNA (siRNA) to c-Jun or by control siRNA. (B1) c-Jun expression was determined by WB analyses. β-Tubulin was used as loading control. (B2) Following siRNA transfection, the cells were stimulated by TNF-α (25 ng/ml; in this part of the study we used a suboptimal concentration of TNF-α in order to facilitate detection of inhibitory effects) for 24 hours. Expression levels of CCL2 and CXCL8 in the supernatants of the cells were determined by ELISA, in the linear range of absorbance. # siRNA to c-Jun has yielded minor increases or reductions in CCL2 and CXCL8 secretion in different experiments (see Results and discussion), and thus overall there was no significant effect on CCL2 and CXCL8 secretion. In all panels, the findings are representatives of n = 3 independent experiments that have shown similar results.

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Derivative Assay, Control, Phospho-proteomics, Western Blot, Transfection, Small Interfering RNA, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: NF-κB is essential in mediating TNF-α-induced release of chemokines by MSCs. (A) Human BM-derived MSCs were stimulated by TNF-α (50 ng/ml) for 15 minutes. The levels of IκBα (the negative regulator of the NF-κB pathway) were determined by WB analyses. GAPDH was used as a loading control throughout. (B) CAFs were generated by culturing MSCs with Tumor CM from MDA-MB-231 (MDA) or MCF-7 breast tumor cells over a prolonged period of time (~30 days). TNF-α (50 ng/ml) was added for the last 24 hours to MSCs + Tumor CM cells and IκBα levels were determined by WB analyses. (C) CAF #1 cells were stimulated for 48 hours by TNF-α (50 ng/ml). IκBα levels were determined by WB analyses. (D) Human BM-derived MSCs were stimulated with TNF-α (50 ng/ml) for 10 minutes. p65 phosphorylation was determined by WB analyses. (E) Human BM-derived MSCs were transiently transfected by siRNA to p65 or by control siRNA. (E1) p65 expression was determined by WB analyses. (E2) Following siRNA transfection, the cells were stimulated by TNF-α (25 ng/ml; a suboptimal concentration of TNF-α in order to facilitate detection of inhibitory effects) for 48 hours. Expression of CCL2 and CXCL8 in the supernatants of the cells was determined by ELISA, in the linear range of absorbance. In all panels, the findings are representatives of n = 3 independent experiments that have shown similar results.

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Derivative Assay, Control, Generated, Phospho-proteomics, Transfection, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: MSCs promote monocyte migration through TNF-α-induced secretion of CCL2. Supernatants (sups) were collected from human BM-derived MSCs that were stimulated by TNF-α (50 ng/ml) or by its vehicle for 24 hours. Thereafter, the sups were incubated with neutralizing antibodies for CCL2 or with nonrelevant isotype-matched control antibody (I.C.) for 30 minutes. The migration of monocytic cells in response to control medium (without chemokine), to recombinant human CCL2 (rhCCL2; 100 ng/ml) or to sups from TNF-α-stimulated or TNF-α nonstimulated MSCs was determined by Boyden chamber migration assays. Cells were counted under a high-power field (HPF). The findings are representatives of n = 3 independent experiments that have shown similar results.

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Migration, Derivative Assay, Incubation, Control, Recombinant

Journal: Anesthesiology

Article Title: Contribution of the Chemokine (C-C Motif) Ligand 2 (CCL2) to Mechanical Hypersensitivity after Surgical Incision in Rats

doi: 10.1097/aln.0b013e3181d3d978

Figure Lengend Snippet: Fig. 1. Time course of mechanical hypersensitivity induced by paw incision after intrathecal treatment with anti-chemokine (C-C motif) ligand 2 (CCL2) immunoglobulin G (IgG) (3 ng, dotted bars; 10 ng, gray bars) or control IgG (10 ng, open bars) was examined acutely for several minutes after treatment beginning 1 day after surgical incision (A) and for several days after single treatment beginning 1 day after surgical incision (B). Withdrawal thresholds to von Frey filaments values are expressed as the median (solid line) and 25th and 75th percentiles of 8–14 rats per group. * P 0.05 for comparisons to control IgG values at each time point or # P 0.05 for comparison between 3 ng anti-CCL2– and 10 ng anti- CCL2–treated values at each time point by Kruskal-Wallis analysis of variance on ranks followed by Dunn post hoc test. Black ar- rowhead indicates time point of intrathecal injection.

Article Snippet: Rats that received plantar incision were intrathecally administered CCL2 neutralizing antibody (goat anti-mouse CCL2/MCP-1/LE 15 l at 200 ng/ml or 15 l at 667 ng/ml for 3 and 10 ng total, respectively) or the same amount of goat anti-mouse immunoglobulin G (IgG) (both antibodies from R&D Systems, Minneapolis, MN).

Techniques: Control, Comparison, Injection

Journal: Anesthesiology

Article Title: Contribution of the Chemokine (C-C Motif) Ligand 2 (CCL2) to Mechanical Hypersensitivity after Surgical Incision in Rats

doi: 10.1097/aln.0b013e3181d3d978

Figure Lengend Snippet: Fig. 2. Representative images of microglial staining with an antibody against ionized calcium-binding adapter molecule 1 (IBA1). Ipsilateral lumbar dorsal spinal cord of rats that underwent sham procedures administered control immunoglobulin G (IgG) (10 ng; A, B, and C) or ipsilateral spinal cord of rats with plantar incision administered 10 ng anti-chemokine (C-C motif) ligand 2 (CCL2) IgG (G, H, and I) or 10 ng control IgG (D, E, and F). Microglial IBA1 immunostaining was increased in the ipsilateral dorsal spinal cord of rats 1 day and 2 days postoperatively (1 dpo and 2 dpo) after plantar incision compared with the levels in rats that underwent sham procedure. A single intrathecal injection of anti-CCL2 IgG (10 ng) administered 1 day after plantar incision reduced the levels of ionized calcium-binding adaptor molecule 1 (IBA1)-immunoreactivity (IR) in the spinal cord of incision rats 24 h after administration (H compared with E) but not 30 min after administration (G compared with D) compared with incision rats that received intrathecal control IgG. Insets depict the morphologic differences in microglia 48 h after incision. Not increased size of microglia in control IgG-treated rats versus anti-CCL2–treated rats (F compared with I). Scale bar in H 150 m and scale bar in I 120 m.

Article Snippet: Rats that received plantar incision were intrathecally administered CCL2 neutralizing antibody (goat anti-mouse CCL2/MCP-1/LE 15 l at 200 ng/ml or 15 l at 667 ng/ml for 3 and 10 ng total, respectively) or the same amount of goat anti-mouse immunoglobulin G (IgG) (both antibodies from R&D Systems, Minneapolis, MN).

Techniques: Staining, Binding Assay, Control, Immunostaining, Injection

Journal: Anesthesiology

Article Title: Contribution of the Chemokine (C-C Motif) Ligand 2 (CCL2) to Mechanical Hypersensitivity after Surgical Incision in Rats

doi: 10.1097/aln.0b013e3181d3d978

Figure Lengend Snippet: Fig. 3. Quantification of ionized calcium-binding adapter molecule 1 (IBA1) immunostaining in ipsilateral (ipsi) (gray bar) and contralateral (contra) (open bar) dorsal spinal cord of sham-operated and incision rats 30 min after intrathecal administration of anti-chemokine (C-C motif) ligand 2 (CCL2) (10 ng) or control immunoglobulin G (IgG) (10 ng) on day 1 postoperatively (1 dpo; A) or 24 h after administration of anti-CCL2 (10 ng) or control IgG (10 ng) on day 2 postoperatively (2 dpo; B). * P 0.05 compared with sham control IgG value within side. # P 0.05 compared with contralateral side within treatment group. P 0.05 compared with incision control IgG-treated values by two-way analysis of variance with Bonferroni post hoc test (n 8 rats per group).

Article Snippet: Rats that received plantar incision were intrathecally administered CCL2 neutralizing antibody (goat anti-mouse CCL2/MCP-1/LE 15 l at 200 ng/ml or 15 l at 667 ng/ml for 3 and 10 ng total, respectively) or the same amount of goat anti-mouse immunoglobulin G (IgG) (both antibodies from R&D Systems, Minneapolis, MN).

Techniques: Binding Assay, Immunostaining, Control

Journal: Anesthesiology

Article Title: Contribution of the Chemokine (C-C Motif) Ligand 2 (CCL2) to Mechanical Hypersensitivity after Surgical Incision in Rats

doi: 10.1097/aln.0b013e3181d3d978

Figure Lengend Snippet: Fig. 4. Representative images of phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) labeling (A, D, and G) and Cd11b (OX42; B, E, and H) in the ipsilateral dorsal spinal cord of sham-operated (A, B, and C) and incision rats treated with 10 ng control immunoglobulin G (IgG) (D, E, and F) or 10 ng anti-chemokine (C-C motif) ligand 2 (CCL2) IgG (G, H, and I) rats 2 days postoperatively (2 dpo) after plantar incision. Rats received a single acute administration of anti-CCL2 IgG (10 ng) on day 1 after plantar incision. High-power confocal images show colocalization of p-p38 MAPK (red) and OX42 (green). Increased levels of cytoplasmic p-p38 MAPK in OX42 immunoreactive microglia of incision rats treated with control IgG compared with sham-operated rats (F compared with C) and incision rats treated with anti-CCL2 IgG (F compared with I). Fluorescent images in panels A, B, D, E, G, and H were inverted in Photoshop (Adobe Systems Inc., San Jose, CA) and converted to grayscale to enhance contrast. Scale bars in G and H 75 m and scale bar in I 5 m.

Article Snippet: Rats that received plantar incision were intrathecally administered CCL2 neutralizing antibody (goat anti-mouse CCL2/MCP-1/LE 15 l at 200 ng/ml or 15 l at 667 ng/ml for 3 and 10 ng total, respectively) or the same amount of goat anti-mouse immunoglobulin G (IgG) (both antibodies from R&D Systems, Minneapolis, MN).

Techniques: Labeling, Control

Journal: Anesthesiology

Article Title: Contribution of the Chemokine (C-C Motif) Ligand 2 (CCL2) to Mechanical Hypersensitivity after Surgical Incision in Rats

doi: 10.1097/aln.0b013e3181d3d978

Figure Lengend Snippet: Fig. 5. Quantification of microglial phosphorylated p38 mitogen-ac- tivated protein kinase immunostaining in ipsilateral (ipsi) (gray bar) and contralateral (contra) (open bar) dorsal spinal cord of sham- operated and incision rats 30 min after intrathecal administration of anti-chemokine (C-C motif) ligand 2 (CCL2) (10 ng) or control immu- noglobulin G (IgG) (10 ng) on day 1 postoperatively (1 dpo; A) or 24 h after administration of anti-CCL2 (10 ng) or control IgG (10 ng) on day 2 postoperatively (2 dpo; B). * P 0.05 compared with sham control IgG value within side. # P 0.05 compared with contralateral side within the treatment group. P 0.05 compared with incision control IgG-treated values by two-way analysis of variance with Bon- ferroni post hoc test (n 8 rats per group).

Article Snippet: Rats that received plantar incision were intrathecally administered CCL2 neutralizing antibody (goat anti-mouse CCL2/MCP-1/LE 15 l at 200 ng/ml or 15 l at 667 ng/ml for 3 and 10 ng total, respectively) or the same amount of goat anti-mouse immunoglobulin G (IgG) (both antibodies from R&D Systems, Minneapolis, MN).

Techniques: Immunostaining, Control

Journal: Anesthesiology

Article Title: Contribution of the Chemokine (C-C Motif) Ligand 2 (CCL2) to Mechanical Hypersensitivity after Surgical Incision in Rats

doi: 10.1097/aln.0b013e3181d3d978

Figure Lengend Snippet: Fig. 6. Correlation between spinal markers of microglial activation and behavioral measures of mechanical hypersensitivity 2 days after surgical incision or sham procedure. The association between num- ber of microglial ionized calcium-binding adaptor molecule 1 immu- noreactive (IBA1-IR) pixels (A) or number of microglia positive for phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) (B) and mechanical paw withdrawal thresholds (g) were ex- amined using Spearman rho nonparametric linear regression analysis. CCL2 chemokine (C-C motif) ligand 2; IgG immunoglobulin G.

Article Snippet: Rats that received plantar incision were intrathecally administered CCL2 neutralizing antibody (goat anti-mouse CCL2/MCP-1/LE 15 l at 200 ng/ml or 15 l at 667 ng/ml for 3 and 10 ng total, respectively) or the same amount of goat anti-mouse immunoglobulin G (IgG) (both antibodies from R&D Systems, Minneapolis, MN).

Techniques: Activation Assay, Binding Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Disruption of tumor-intrinsic PGAM5 increases anti-PD-1 efficacy through the CCL2 signaling pathway

doi: 10.1136/jitc-2024-009993

Figure Lengend Snippet: PGAM5 reduced CCL2 secretion in HCC cells by downregulating DRP1. ( A ) Schematic diagram of the in vitro co-culture system for Huh7 shctrl or shPGAM5 cells and phorbol-12-myristate-13-acetate-induced THP-1 cells. ( B ) Quantitative PCR analysis of macrophage polarization maker genes of THP-1 cells co-cultured with indicated HCC cells. ( C ) cytokine antibody array incubated with culture medium of Huh7 shctrl or shPGAM5 cells. ( D ) Quantitative PCR analysis of macrophage M1/M2 polarization maker genes of THP-1 in the co-culture system, culture medium of Huh7 shPGAM5 cells were supplemented with recombinant CCL2 and HCCLM3 PGAM5 OE cells were supplemented with neutralizing-CCL2 antibody. ( E ) TOP20 GO enrichment of differentially expressed proteins in Huh7 shPGAM5 cells compared with shctrl cells. ( F ) Volcano plot of significantly differentially expressed proteins in Huh7 shPGAM5 cells compared with shctrl cells. ( G ) Western blot analysis of protein expression of PGAM5 and mitochondria dynamic proteins DRP1, MFN1, MFN2, OPA1 in HCC cells. ( H ) Western blot analysis of DRP1 overexpression efficiency in Huh7 shPGAM5 cells by lenti-virus infection. ( I ) Concentration of CCL2 in the supernatant of indicated HCC cells was measured by ELISA. ( J ) Western blot analysis of DRP1 silencing efficiency in HCCLM3 PGAM5-OE cells by transient transfection of SiRNA. ( K ) Concentration of CCL2 in the supernatant of indicated HCC cells was measured by ELISA kit. *p<0.05, **p<0.01, and ***p<0.001, ****p<0.0001, ns: not significant. HCC, hepatocellular carcinoma; IHC, immunohistochemistry; UMAP, uniform manifold approximation and projection.

Article Snippet: For cytokine rescue or antibody neutralizing, recombinant human CCL2 protein (CSB-EP004783HUb1; cusabio) at 400 ng/mL or anti-CCL2 neutralizing antibody (Cat. MAB679; R&D Systems) at 20 μg/mL was supplemented in the co-culture system at day 3.

Techniques: In Vitro, Co-Culture Assay, Real-time Polymerase Chain Reaction, Cell Culture, Ab Array, Incubation, Recombinant, Western Blot, Expressing, Over Expression, Virus, Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transfection, Immunohistochemistry

Journal: Journal for Immunotherapy of Cancer

Article Title: Disruption of tumor-intrinsic PGAM5 increases anti-PD-1 efficacy through the CCL2 signaling pathway

doi: 10.1136/jitc-2024-009993

Figure Lengend Snippet: PGAM5 deficiency inhibits mitochondria fission and downstream mtDNA-TLR9-NF-κB-CCL2 pathway. ( A ) Mitochondria morphology of HCC cells with PGAM5 knock-down or overexpression indicated by mitotracker. ( B ) Confocal images of cytosolic mtDNA stained with picogreen (green) and mitochondria with mitotracker (red) in HCC cells as indicated. ( C ) Cytosolic mtDNA quantified by quantitative PCR in HCC cells with PGAM5 knock-down or overexpression. ( D ) Co-localization of TLR9 (red) and mtDNA (green) in Huh7 shctrl and shPGAM5 cells as indicated by confocal images. ( E ) upper: schematic of three NF-κB-binding sites in the promoter region of the CCL2. Bottom: chromatin immunoprecipitation analysis of NF-κB-binding sites in CCL2 promoter region in Huh7 cells. Anti-Histone3 lysates were used as positive control. ( F ) Western blot analysis of NF-κB in the cytoplasm and nuclear in Huh7 shctrl and shPGAM5 cells. ( G ) NF-κB distribution in cytoplasm and nuclear in Huh7 cells treated as indicated. ( H ) Concentration of CCL2 in the supernatants of Huh7 cells treated as indicated. *p<0.05, **p<0.01, and ***p<0.001, ****p<0.0001, ns: not significant. HCC, hepatocellular carcinoma.

Article Snippet: For cytokine rescue or antibody neutralizing, recombinant human CCL2 protein (CSB-EP004783HUb1; cusabio) at 400 ng/mL or anti-CCL2 neutralizing antibody (Cat. MAB679; R&D Systems) at 20 μg/mL was supplemented in the co-culture system at day 3.

Techniques: Knockdown, Over Expression, Staining, Real-time Polymerase Chain Reaction, Binding Assay, Chromatin Immunoprecipitation, Positive Control, Western Blot, Concentration Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Disruption of tumor-intrinsic PGAM5 increases anti-PD-1 efficacy through the CCL2 signaling pathway

doi: 10.1136/jitc-2024-009993

Figure Lengend Snippet: Schematic mechanism for the effect of tumor-intrinsic PGAM5 on tumor-associated macrophages. Mitochondria in hepatocellular carcinoma cells are in a status of hyperfission. PGAM5 deficiency promotes TRIM28 bind to DRP1 and increase DRP1 ubiquitination, which accelerates DRP1 degradation by proteasome. DRP1 downregulation inhibits mitochondria fission. The leakage of mtDNA was reduced, downstream TLR9-NF-κB-CCL2 axis is deactivated. The attenuation of CCL2 secretion facilitates TAM repolarization to M1 phenotype and potentiates PD-1 antibody-mediated activation of CD8 + T cells. PD-1, programmed cell death protein-1; TAM, tumor-associated macrophage; TGF, transforming growth factor; TNF, tumor necrosis factor.

Article Snippet: For cytokine rescue or antibody neutralizing, recombinant human CCL2 protein (CSB-EP004783HUb1; cusabio) at 400 ng/mL or anti-CCL2 neutralizing antibody (Cat. MAB679; R&D Systems) at 20 μg/mL was supplemented in the co-culture system at day 3.

Techniques: Ubiquitin Proteomics, Activation Assay

Journal: The Journal of Neuroscience

Article Title: Peripheral Nerve Injury Alters Blood–Spinal Cord Barrier Functional and Molecular Integrity through a Selective Inflammatory Pathway

doi: 10.1523/JNEUROSCI.1642-11.2011

Figure Lengend Snippet: Experimental protocols for immune modulation of BSCB integrity

Article Snippet: Rat MCP-1 neutralizing antibody , Nerve-injured rats , 3 d (day 0 to day 3) , Intrathecal (osmotic pumps) , 3 μg (PeproTech).

Techniques: Recombinant, Derivative Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CPEB3 inhibits epithelial-mesenchymal transition by disrupting the crosstalk between colorectal cancer cells and tumor-associated macrophages via IL-6R/STAT3 signaling

doi: 10.1186/s13046-020-01637-4

Figure Lengend Snippet: CPEB3 modulates CCL2 secretion in CRC cell supernatants to regulate TAM polarization (a) We measured the expression of the respective inflammatory cytokines in cell culture supernatants of HCT116-Ctrl/CPEB3 cells by ProcartaPlex combinable panels; error bars, SEM. (b) We measured the expression of the respective inflammatory cytokines in the supernatants of LoVo-shCtrl/shCPEB3 cells by ProcartaPlex combinable panels; error bars, SEM. (c) THP-1 macrophages were co-cultured with LoVo-shCtrl/shCPEB3 with or without CCL2-neutralizing antibody (1 μg/mL) for 24 h. Flow cytometry was used to explore the surface expression of CD86 and CD163 in the differentiated macrophages; error bars, SEM. (d) THP-1 macrophages were co-cultured with RKO-shCtrl/shCPEB3 cells with or without a CCL2-neutralizing antibody (1 μg/mL) for 24 h. Flow cytometry was used to explore the surface expression of CD86 and CD163 in the differentiated macrophages. Error bars, SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

Article Snippet: THP-1 macrophages were co-cultured with stably transfected CRC cells along with or without neutralizing antibodies to CCL2 (anti-CCL2; R&D Systems).

Techniques: Expressing, Cell Culture, Flow Cytometry