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: High-dimensional analyses reveal both unique and shared macrophage genes and functions after force application. (A) Bar chart of the relative proportions of clusters from two groups. (B) Heatmap of the top 30 differentially expressed genes in control only, OTM only, and overlapping populations. (C) Heatmap of the top 15 differentially expressed genes in Ccr2 cluster between two groups. (D) Pathway analysis (gProfiler) of commonly and differentially expressed genes in Ccr2 cluster. (E) Representative immunofluorescence staining and quantification of CD68 + CCR2 + macrophages in the human periodontal tissue. Scale bar = 20μm. Values are mean ± SD. n = 5. **P < 0.01.

Article Snippet: Subsequently, samples were blocked for 30min with 10% BSA at room temperature and incubated with anti-CD68 (1:200, ab955; Abcam) and CCR2 antibodies (1:100, bs-0562R; Bioss) overnight at 4°C to detect CCR2 + macrophages.

Techniques: 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: 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: Subsequently, samples were blocked for 30min with 10% BSA at room temperature and incubated with anti-CD68 (1:200, ab955; Abcam) and CCR2 antibodies (1:100, bs-0562R; Bioss) overnight at 4°C to detect CCR2 + macrophages.

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: Subsequently, samples were blocked for 30min with 10% BSA at room temperature and incubated with anti-CD68 (1:200, ab955; Abcam) and CCR2 antibodies (1:100, bs-0562R; Bioss) overnight at 4°C to detect CCR2 + macrophages.

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

Journal: Cancer Medicine

Article Title: Combination therapy with bevacizumab and a CCR2 inhibitor for human ovarian cancer: An in vivo validation study

doi: 10.1002/cam4.5674

Figure Lengend Snippet: Combination therapy with BEV and a high‐dose CCR2i suppressed tumor growth by inhibiting the CCR2B‐MAPK pathway. (A) Representative image of the mouse blood vessel structure on the tumor surface in each treatment for the third PDX. α‐smooth muscle actin is stained in green fluorescence. The white bar shows a length of 1 mm, and the broken white lines indicate the tumor edges. (B) Left box plot: density of xenograft‐derived microvessel in each treatment for the third PDX, *: a significant difference. Right images: representative human CD31 staining in each treatment for the first PDX. The black bar shows a length of 250 μm. (C) CCR2B expression. Representative images of CCR2B staining in each treatment for the first PDX. Left top: low‐power field (× 40). The black bar shows a length of 1 mm. Left bottom: high‐power field (× 100). The black bar shows a length of 100 μm. Right box plot: RT–PCR analysis of CCR2B mRNA expression. *: a significant difference.

Article Snippet: Membranes were blocked in 5% non‐fat dry milk in TBS‐T buffer (0.1% Tween‐20 in Tris‐buffered saline) for 1 h at room temperature and then exposed to primary antibodies against CCR2A (1:1000 dilution, Cat# 16153‐1‐AP, RRID:AB_2262945; Proteintech), and CCR2B (1:1000 dilution; Cat# 16154‐1‐AP, RRID:AB_2878224; Proteintech), p44/42 MAPK (Erk1/2, 1:1000 dilution, Cat# 9102, RRID:AB_330744; Cell Signaling Technology), phosphor‐p44/42 MAPK (Erk1/2 Thr202/Tyr204, 1:1000 dilution, Cat# 9101, RRID:AB_331646; Cell Signaling Technology), and anti‐actin (1:1000 dilution, Cat# MAB1501, RRID:AB_2223041; Millipore) in 5% milk/TBS‐T or 5%BSA/TBS‐T overnight at 4 °C.

Techniques: Staining, Fluorescence, Derivative Assay, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Cancer Medicine

Article Title: Combination therapy with bevacizumab and a CCR2 inhibitor for human ovarian cancer: An in vivo validation study

doi: 10.1002/cam4.5674

Figure Lengend Snippet: CCR2i suppressed the growth of tumors resistant to combination therapy with BEV and the CCR2i by direct inhibition of the CCR2‐MAPK pathway. (A) Left bar graph: density of xenograft‐derived microvessels in each treatment for the second PDX. Data are shown as the means ± standard error of triplicate assays. Right pictures: representative images of CD31 staining in each treatment for the second PDX. (B) Left images: representative pictures of CCR2B staining in each treatment for the second PDX. The black bar shows a length of 5 mm. Right graph: Results of an RT–PCR analysis of the CCR2B mRNA expression. Data are shown as the means ± the standard error and *: a significant difference. (C) Left images: immunoblot of CCR2A, CCR2B, ERK, phosphorylated ERK (pERK), and actin expression in each treatment (left four: BEV, right four: BEV + CCR2i) for eight samples from the second PDX. Right graph: normalized CCR2A, CCR2B, ERK, and phosphorylated ERK (pERK) expression in each treatment for the second PDX. Data are shown as the means ± the standard error. *: a significant difference.

Article Snippet: Membranes were blocked in 5% non‐fat dry milk in TBS‐T buffer (0.1% Tween‐20 in Tris‐buffered saline) for 1 h at room temperature and then exposed to primary antibodies against CCR2A (1:1000 dilution, Cat# 16153‐1‐AP, RRID:AB_2262945; Proteintech), and CCR2B (1:1000 dilution; Cat# 16154‐1‐AP, RRID:AB_2878224; Proteintech), p44/42 MAPK (Erk1/2, 1:1000 dilution, Cat# 9102, RRID:AB_330744; Cell Signaling Technology), phosphor‐p44/42 MAPK (Erk1/2 Thr202/Tyr204, 1:1000 dilution, Cat# 9101, RRID:AB_331646; Cell Signaling Technology), and anti‐actin (1:1000 dilution, Cat# MAB1501, RRID:AB_2223041; Millipore) in 5% milk/TBS‐T or 5%BSA/TBS‐T overnight at 4 °C.

Techniques: Inhibition, Derivative Assay, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot

Journal: Pediatrics International

Article Title: Imbalance of peroxisome proliferator-activated receptor gamma and adiponectin predisposes Kawasaki disease patients to developing atherosclerosis

doi: 10.1111/j.1442-200x.2010.03160.x

Figure Lengend Snippet: Fig. 1 Plot of the respective gene expression in Kawasaki disease (KD) patients and normal control (NC) subjects. The patients with coronary artery lesions are indicated as closed circles. The values represent the ratios of target gene mRNA to b-actin mRNA (target/b-actin). Peroxisome proliferator-activated receptor g (PPARg) mRNA expression was significantly upregulated in both acute and convalescent KD patients (acute KD, P = 0.0008; convalescent KD, P = 0.014). The CCR2 mRNA expression levels were significantly elevated in convalescent KD patients (P = 0.0003). The monocyte chemoattractant protein-1 (MCP-1) mRNA levels were elevated in convalescent KD patients, but this difference was not statistically significant.

Article Snippet: Real-time PCR was performed with an appropriate dilution of cDNA using the Applied Biosystems 7500 Real-Time PCR System sequence detector and the TaqMan Gene Expression Assays kit (PPARg, Hs01115513_m1; MCP-1, Hs00704702_s1; CCR2, Hs00234140_m1; Applied Biosystems) according to the manufacturer’s instructions.

Techniques: Gene Expression, Control, Expressing