recombinant proteins rs 504393 tocris  (Tocris)

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: 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