Journal: Cell reports

Article Title: Bone Marrow Mesenchymal Stromal Cell-Derived Periostin Promotes B-ALL Progression by Modulating CCL2 in Leukemia Cells.

doi: 10.1016/j.celrep.2019.01.034

Figure Lengend Snippet: Figure 6. CCL2 Promotes the Expression of POSTN in BM-MSCs via STAT3 Activation (A) qRT-PCR analysis of the levels of POSTN, c-JUN, RUNX1, RUNX2, STAT3, and YY1 in BM-MSCs and rmCCL2-treated BM-MSCs (n = 3 per group). (B) Western blot analysis of the levels of POSTN, phosphorylated STAT3 (p-STAT3), and STAT3 in BM-MSCs isolated from mice with or without B-ALL. (C) Western blot analysis of the levels of POSTN, p-STAT3, and STAT3 in BM-MSCs and rmCCL2-treated BM-MSCs pre-incubated with or without RS504393. (D) Western blot analysis of the levels of p-STAT3 and STAT3 in BM-MSCs and rmCCL2-treated BM-MSCs pre-incubated with or without RS504393. (E) Western blot analysis of the levels of POSTN, p-STAT3, and STAT3 in BM-MSCs and co-cultured BM-MSCs pre-incubated with or without RS504393. (F) Western blot analysis of the levels of p-STAT3 and STAT3 in mouse BM-MSCs co-cultured with L1210-GFP-Luc-sh-ctrl cells or L1210-GFP-Luc-sh-CCL2 cells. (G) Western blot analysis of the levels of p-STAT3 and STAT3 in BM-MSCs transfected with siRNAs to knock down STAT3. (H and I) qRT-PCR assay of the mRNA levels of STAT3 (H) and POSTN (I) in mouse BM-MSCs and rmCCL2-treated and siRNA-transfected BM-MSCs (n = 3 per group). (J) Western blot analysis of the levels of POSTN, p-STAT3, and STAT3 in mouse BM-MSCs and rmCCL2-treated and siRNA-transfected BM-MSCs. (K) Analysis of the effect of STAT3 knockdown in BM-MSCs on CCL2-promoted adhesion of L1210-GFP-Luc cells to mouse BM-MSCs (n = 6 per group). The graphs show mean ± SD. Significance is indicated as follows: **p < 0.01; ***p < 0.001; ns, no significance.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-POSTN Adipogen Cat# AG-20B-0033 Rabbit monoclonal anti-GFP Abcam Cat# ab183734 Mouse monoclonal anti-MCP-1 Cell Signaling Technology Cat# 2029 Rabbit monoclonal anti-Intergrin Linked ILK Abcam Cat# ab52480 Rabbit monoclonal anti-Phospho-NF-kB p65 Cell Signaling Technology Cat# 3033T Rabbit monoclonal anti-NF-kB p65 Cell Signaling Technology Cat# 8242 Rabbit monoclonal anti-Phospho-STAT3 Cell Signaling Technology Cat# 9145 Mouse monoclonal anti-STAT3 Cell Signaling Technology Cat# 9132 Rabbit monoclonal anti-AKT Cell Signaling Technology Cat# 9272 Rabbit monoclonal anti-Phospho-FAK Cell Signaling Technology Cat# 3281 Mouse monoclonal anti-Phospho-JNK Cell Signaling Technology Cat# 9255 Rabbit monoclonal anti-JNK Cell Signaling Technology Cat# 9252 Rabbit monoclonal anti-Phospho-AKT Cell Signaling Technology Cat# 9271 Anti-Integrin aVb3 antibody Millipore/Merck Cat# MAB1876-Z Anti-Integrin aVb5 antibody Millipore/Merck Cat# MAB1961 Rabbit monoclonal anti-Ki67 Abcam Cat# ab15580 Rabbit monoclonal anti-ERK Cell Signaling Technology Cat# 4695 Rabbit monoclonal anti-Phospho-ERK Cell Signaling Technology Cat# 3211 Mouse monoclonal anti-FAK BD Biosciences Cat# 610087 Rabbit monoclonal anti-b-actin ABclonal Cat# AC026 Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP Thermo Fisher Scientific Cat# 31460 Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP Thermo Fisher Scientific Cat# 31430 Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546 Thermo Fisher Scientific Cat# A10040 Biological Samples Human bone marrow tissue samples The First Affiliated Hospital of Hunan Normal University N/A Human serum samples The First Affiliated Hospital of Hunan Normal University N/A Chemicals, Peptides, and Recombinant Proteins CCR2 Chemokine receptor anagonist RS504393 TOCRIS Cat# 2517 ILK inhibitor Cpd 22 Millipore/Merck Cat# 407331 BAY11-7082 MedChemExpress Cat# HY-13453 PD98059 MedChemExpress Cat# HY-12028 Human Periostin/OSF-2 DuoSet ELISA R&D Cat# DY3548B Human CCL2/MCP-1 DuoSet ELISA R&D Cat# DY279 Recombinant Human Periostin/OSF-2 Protein, CF R&D Cat# 3548-F2 Recombinant Mouse Periostin/OSF-2 Protein, CF R&D Cat# 2955-F2 Recombinant Mouse CCL2/JE/MCP-1 Protein R&D Cat# 479-JE-050 (Continued on next page) e1 Cell Reports 26, 1533–1543.e1–e4, February 5, 2019

Techniques: Expressing, Activation Assay, Quantitative RT-PCR, Western Blot, Isolation, Incubation, Cell Culture, Transfection, Knockdown

Journal: Molecular Pain

Article Title: Nuclear factor kappa B regulated monocyte chemoattractant protein-1/chemokine CC motif receptor-2 expressing in spinal cord contributes to the maintenance of cancer-induced bone pain in rats

doi: 10.1177/1744806918788681

Figure Lengend Snippet: Intrathecal injection of CCR2 inhibitor (RS504393) reduced bone cancer pain and affected the production of inflammation factors. (a) CCR2 antagonist (RS504393) at a lower dose (50 μg) had mild effect on Walker-256 cell inoculation-induced pain hypersensitivity, whereas the antagonist at a higher dose (100 μg) reversed inoculation-induced mechanical allodynia for more than 4 h. *p < 0.05, **p < 0.01, vs. control serum. n = 12 rats per group. (b–d) CCR2 antagonist (RS504393) at a lower dose (25 μg) had a slight effect on inflammation cytokine and at a higher dose (50 μg) had a much more dramatic effect. *p < 0.05, **p < 0.01, vs. control serum. n = 4 rats per group. CIBP: cancer-induced bone pain; DMSO: dimethyl sulfoxide; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; TNF-α: tumor necrosis factor-α; IL-4: interleukin-4; IFN-γ: interferon-γ.

Article Snippet: RS504393, an effective and selective antagonist of CCR2, was purchased from Boster (Shanghai, China).

Techniques: Injection, Control

Journal: Respiratory Research

Article Title: Loss of the adhesion G-protein coupled receptor ADGRF5 in mice induces airway inflammation and the expression of CCL2 in lung endothelial cells

doi: 10.1186/s12931-019-0973-6

Figure Lengend Snippet: Suppressive effect of a CCR2 antagonist on the expression of S100a8 , S100a9 , Slc26a4 , and Il5 in Adgrf5 −/− lungs. Two-week-old Adgrf5 −/− mice were administered RS504393 (2 mg/kg body weight) or vehicle once daily via subcutaneous injection for 8 days. The mRNA expression of Ccl2 , S100a8 , S100a9 , Saa3 , Slc26a4 , Clca1 , Tgfb1 , Il5 , and Il13 was analyzed by qPCR using total RNA isolated from post-lavage lungs of injected mice and non-injected WT mice at 3 weeks of age. The data were normalized to Gapdh levels and are expressed as values relative to those from corresponding vehicle-treated Adgrf5 −/− mice. Values are presented as the mean ± SEM ( n = 3–4). * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: RS504393 (Cayman Chemical, Ann Arbor, MI, USA) was dissolved in dimethylformamide at 10 mg/ml and then diluted to 0.4 mg/ml with a 0.9% sterile NaCl solution.

Techniques: Expressing, Injection, Isolation