ccr2 tango  (Addgene inc)

Journal: Pharmacological research

Article Title: α 1 -adrenoceptor ligands inhibit chemokine receptor heteromerization partners of α 1B/D -adrenoceptors via interference with heteromer formation.

doi: 10.1016/j.phrs.2023.106730

Figure Lengend Snippet: Fig. 1. Ligand free α1b-AR facilitates CCR2-mediated activation of Gαi1. A/B. HEK293T cells were transfected with CCR2, Gαi1-Rluc8, Gβ3, and Gγ9-GFP2 together with pcDNA3 or α1b-AR as indicated in the figure. Forty-eight hours after transfection, cells were used for BRET assays and flow cytometry. A. Flow cytometry for the detection of HA-CCR2 with anti-HA (left) and of FLAG-α1b-AR with anti-FLAG (right). Red line: cells transfected with G protein biosensors, HA- CCR2 plus pcDNA3. Green line: cells transfected with G protein biosensors, HA-CCR2 plus FLAG-α1b-AR. Grey area: unstained cells. B. BRET to measure Gαi1 activation. Cells transfected with CCR2 plus pcDNA3 (open circles) or with CCR2 plus α1b-AR (grey squares) were stimulated with CCL2 and BRET measured. BRET change: BRET in the presence minus BRET in the absence of CCL2. Data are mean ± SE, n = 4. * : p < 0.05 for CCR2 plus pcDNA3 vs. CCR2 plus α1b-AR. C. Flow cytometry for the detection of FLAG-CCR2-Tango with anti-FLAG (left) and of HA-α1b-AR with anti-HA (right). HTLA cells were transfected with FLAG-CCR2-Tango plus pcDNA3 (green line) or HA-α1b-AR (red line). Grey area: unstained cells. D. CCR2 PRESTO-Tango β-arrestin-2 recruitment assays. HTLA cells were transfected as in C. and stimulated with various concentrations of CCL2. Data are mean ± SE, n = 3. RLU, fold change: relative luminescence units (RLU) expressed as fold change in RLU over cells not exposed to CCL2.

Article Snippet: FLAG-CCR2-Tango was generated by insertion of CCR2 cDNA in the empty TANGO vector. α1b-AR and CCR2 cDNA were fused at Age I and Xba I sites with either RlucII or EYFP at C-terminus to form α1b-AR-RlucII and CCR2-EYFP, as described [14]. β-arrestin-2 mYFP was from Addgene (#36917) deposited by the laboratory of Dr. Robert Lefkowitz.

Techniques: Activation Assay, Transfection, Flow Cytometry

Journal: Pharmacological research

Article Title: α 1 -adrenoceptor ligands inhibit chemokine receptor heteromerization partners of α 1B/D -adrenoceptors via interference with heteromer formation.

doi: 10.1016/j.phrs.2023.106730

Figure Lengend Snippet: Fig. 2. Depletion of α1B-AR inhibits CCR2-mediated Gαi signaling. A. THP-1 (open bars) and THP-1ADRA1BKO (grey bars) cells were incubated with vehicle (ctrl.) or 10 μM forskolin (FSK) and cAMP concentrations in cell lysates measured. Data are mean ± SE, n = 3. B. Forskolin-treated THP-1 (open bars) and THP- 1ADRA1BKO (grey bars) cells were stimulated with 100 nM CCL2 or CCL1 for 15 min at 37 ℃ and cAMP concentrations in cell lysates measured. cAMP concen trations (pmol/mL) are expressed as %inhibition of cAMP concentrations in cells not exposed to chemokines. Data are mean ± SE, n = 4. * : p < 0.05 THP-1 vs. THP1_ADRA1BKO cells. C. Flow cytometry to analyze cell surface receptor expression in THP-1 cells after siRNA gene silencing. Gray: unstained cells; red: cells incubated with NT siRNA; green: cells incubated with α1B-AR siRNA. D. Median fluorescence intensities (MFI) of the receptor signals in cells as in C. MFI is expressed as % of MFI in cells incubated with NT siRNA. * ; p < 0.05 vs.cells incubated with NT siRNA. Data are mean ± SE, n = 4. E. THP-1 cells after incubation with NT siRNA (white bars) or α1B-AR siRNA (grey bars) were incubated with vehicle (ctrl.) or 10 μM forskolin (FSK) and cAMP concentrations in cell lysates measured. Data are mean ± SE, n = 4. F. Forskolin-treated THP-1 cells, as in E., cells were stimulated with 100 nM CCL2 or CCL1 for 15 min at 37 ℃ and cAMP concentrations in cell lysates measured. cAMP concentrations (pmol/mL) are expressed as %inhibition of cAMP concentrations in cells not exposed to chemokines. Data are mean ± SE, n = 4. * : p < 0.05 cells incubated with NT siRNA vs. cells incubated with α1B-AR siRNA.

Article Snippet: FLAG-CCR2-Tango was generated by insertion of CCR2 cDNA in the empty TANGO vector. α1b-AR and CCR2 cDNA were fused at Age I and Xba I sites with either RlucII or EYFP at C-terminus to form α1b-AR-RlucII and CCR2-EYFP, as described [14]. β-arrestin-2 mYFP was from Addgene (#36917) deposited by the laboratory of Dr. Robert Lefkowitz.

Techniques: Incubation, Inhibition, Flow Cytometry, Cell Surface Receptor Assay, Expressing, Fluorescence

Journal: Pharmacological research

Article Title: α 1 -adrenoceptor ligands inhibit chemokine receptor heteromerization partners of α 1B/D -adrenoceptors via interference with heteromer formation.

doi: 10.1016/j.phrs.2023.106730

Figure Lengend Snippet: Fig. 3. α1b-AR ligands reduce the heteromerization propensity between α1b-AR and CCR2. HEK293T cells were co-transfected with a fixed amount of α1b-AR-RlucII and increasing amounts of CCR2-EYFP. 48 h after transfection, cells were treated with vehicle (ctrl.), phenylephrine (PE, 10 μM) or phentol amine (PT, 10 μM) for 5 min at 37ºC before measuring BRET. A. Representative measurements from a titration BRET experiment. B. BRETmax values. Data are mean ± SE, n = 4. C. BRET50 values. Data are mean ± SE, n = 4. * : p < 0.05 vs. ctrl.

Article Snippet: FLAG-CCR2-Tango was generated by insertion of CCR2 cDNA in the empty TANGO vector. α1b-AR and CCR2 cDNA were fused at Age I and Xba I sites with either RlucII or EYFP at C-terminus to form α1b-AR-RlucII and CCR2-EYFP, as described [14]. β-arrestin-2 mYFP was from Addgene (#36917) deposited by the laboratory of Dr. Robert Lefkowitz.

Techniques: Transfection, Titration

Journal: Pharmacological research

Article Title: α 1 -adrenoceptor ligands inhibit chemokine receptor heteromerization partners of α 1B/D -adrenoceptors via interference with heteromer formation.

doi: 10.1016/j.phrs.2023.106730

Figure Lengend Snippet: Fig. 4. α1b-AR ligands regulate CCR2 mediated activation of Gαi1 from the α1b-AR:CCR2 heteromer. A-F. HEK293T cells were transfected with Gαi1-Rluc8, Gβ3, and Gγ9-GFP2 together with pcDNA3, CCR2 or α1b-AR as indicated. Forty-eight hours after transfection, cells were used for BRET assays. BRET change: BRET in the presence minus BRET in the absence of receptor ligands. Data are mean ± SE, n = 4 or 5. * : p < 0.05 vs. vehicle. A. Cells transfected as indicated were exposed to various concentrations of phenylephrine (PE) or phentolamine (PT). B. Cells transfected with CCR2 plus pcDNA3 were stimulated with various concentrations of CCL2 plus vehicle or 10 μM PE. C. Cells transfected with CCR2 plus α1b-AR were stimulated various concentrations of CCL2 plus vehicle or 10 μM PE. D. Cells transfected with CCR2 plus α1b-AR were stimulated with various concentrations of CCL2 plus vehicle or 1 μM norepinephrine (NE). E. Cells transfected with CCR2 plus pcDNA3 were stimulated various concentrations of CCL2 plus vehicle or 10 μM PT. F. Cells transfected with CCR2 plus α1b-AR were stimulated various con centrations of CCL2 plus vehicle or 10 μM PT. G. Forskolin-treated THP-1 cells were stimulated with various concentrations of CCL2 in the presence of vehicle (open circles), 1 μM PE (grey squares) or 1 μM PT (grey triangles) for 15 min at 37 ℃ and cAMP concentrations in cell lysates measured. cAMP concentrations (pmol/mL) are expressed as % cells not exposed to CCL2. Data are mean ± SE, n = 4. * : p < 0.05 vs. vehicle. H. Forskolin-treated THP-1 cells were stimulated with 10 nM CCL2 in the presence of vehicle or 100 nM NE for 15 min at 37 ℃ and cAMP concentrations in cell lysates measured. cAMP concentrations (pmol/mL) are expressed as % of cells not exposed to CCL2 or NE. Data are mean ± SE, n = 4. * : p < 0.05 vs. vehicle.

Article Snippet: FLAG-CCR2-Tango was generated by insertion of CCR2 cDNA in the empty TANGO vector. α1b-AR and CCR2 cDNA were fused at Age I and Xba I sites with either RlucII or EYFP at C-terminus to form α1b-AR-RlucII and CCR2-EYFP, as described [14]. β-arrestin-2 mYFP was from Addgene (#36917) deposited by the laboratory of Dr. Robert Lefkowitz.

Techniques: Activation Assay, Transfection

Journal: Pharmacological research

Article Title: α 1 -adrenoceptor ligands inhibit chemokine receptor heteromerization partners of α 1B/D -adrenoceptors via interference with heteromer formation.

doi: 10.1016/j.phrs.2023.106730

Figure Lengend Snippet: Fig. 5. Activation of α1b-AR enhances β-arrestin-2 recruitment to CCR2. A/B. CCR2 PRESTO-Tango β-arrestin-2 recruitment assays. HTLA cells were transfected with FLAG-CCR2- Tango plus pcDNA3 or HA-α1b-AR, as in Fig. 1 C/D. RLU, fold change: relative lumines cence units (RLU) expressed as fold change in RLU over cells not exposed to receptor ligands. A. Cells transfected as indicated were stimu lated with various concentrations of PE or PT. Data are mean ± SE, n = 4. * : p < 0.05 vs. unstimulated cells. B. Cells transfected with CCR2-Tango plus α1b-AR were stimulated with various concentrations of CCL2 in the presence of vehicle, 1 μM PE or 1 μM PT. Data are mean ± SE, n = 4. * : p < 0.05 vs. cells stimulated with CCL2 plus vehicle. C/D. BRET assay for β-arrestin-2 recruitment. HEK293T cells were transfected with CCR2-RLuc and β-arrestin-2- YFP with (D) or without (C) α1b-AR. Cells were treated with various concentrations of CCL2 plus vehicle, 10 μM PE, PT or 100 nM INCB3284 before BRET measurements. Data are mean ± SE, n = 4. * : p < 0.05 vs. vehicle.

Article Snippet: FLAG-CCR2-Tango was generated by insertion of CCR2 cDNA in the empty TANGO vector. α1b-AR and CCR2 cDNA were fused at Age I and Xba I sites with either RlucII or EYFP at C-terminus to form α1b-AR-RlucII and CCR2-EYFP, as described [14]. β-arrestin-2 mYFP was from Addgene (#36917) deposited by the laboratory of Dr. Robert Lefkowitz.

Techniques: Activation Assay, Transfection, Bioluminescence Resonance Energy Transfer

Journal: Pharmacological research

Article Title: α 1 -adrenoceptor ligands inhibit chemokine receptor heteromerization partners of α 1B/D -adrenoceptors via interference with heteromer formation.

doi: 10.1016/j.phrs.2023.106730

Figure Lengend Snippet: Fig. 7. Agonist binding to α1B/D-AR enhances CCL2-induced internalization of CCR2. A. THP-1 cells were incubated with 100 nM CCL2 (left) or 100 nM CCL2 plus 1 μM phenylephrine (PE, right) at 37 ◦C for various time periods (0–45 min, color coded as indicated). Cell surface CCR2 expression was analyzed by flow cytometry. Grey areas: unstained cells. B. Quantification of CCR2 expression from n = 4 independent experiments, as in A. Open circles: cells stimulated with CCL2. Grey squares: cells stimulated with CCL2 plus PE. CCR2 expression (% ctrl.): median fluorescence intensity (MFI) in percent of the MFI measured at 0 min (=100%). Data are mean ± SE. * : p < 0.05 CCL2 vs. CCL2 plus PE.

Article Snippet: FLAG-CCR2-Tango was generated by insertion of CCR2 cDNA in the empty TANGO vector. α1b-AR and CCR2 cDNA were fused at Age I and Xba I sites with either RlucII or EYFP at C-terminus to form α1b-AR-RlucII and CCR2-EYFP, as described [14]. β-arrestin-2 mYFP was from Addgene (#36917) deposited by the laboratory of Dr. Robert Lefkowitz.

Techniques: Binding Assay, Incubation, Expressing, Flow Cytometry, Fluorescence

Journal: Pharmacological research

Article Title: α 1 -adrenoceptor ligands inhibit chemokine receptor heteromerization partners of α 1B/D -adrenoceptors via interference with heteromer formation.

doi: 10.1016/j.phrs.2023.106730

Figure Lengend Snippet: Fig. 6. Agonist binding to α1B/D-AR induces co-internalization α1B/D-AR and their chemokine receptor heteromerization partners. A. THP-1 cells were incubated with vehicle (ctrl., top), 10 μM phenylephrine (PE, center) or 10 μM phentolamine (PT, bottom) for 30 min at 37 ◦C and cell surface expression of in dividual receptors visualized by PLA. Images show merged 4′,6-diamidino-2-phenylindole (DAPI, nuclear counterstain) and PLA signals (red, λexcitation/emission 598/634 nm) acquired from z-stack images (n = 10; thickness 0.5 µm, bottom to top) and are representative of n = 3 independent experiments. Scale bar, 10 µm. B- G. Quantification of PLA signals for α1B-AR (B), α1D-AR (C), CCR1 (D), CCR2 (E), CCR8 (F) and CXCR4 (G) from n = 3 experiments. Data are mean ± SE, n = 3. * : p < 0.05 vs. ctrl.

Article Snippet: FLAG-CCR2-Tango was generated by insertion of CCR2 cDNA in the empty TANGO vector. α1b-AR and CCR2 cDNA were fused at Age I and Xba I sites with either RlucII or EYFP at C-terminus to form α1b-AR-RlucII and CCR2-EYFP, as described [14]. β-arrestin-2 mYFP was from Addgene (#36917) deposited by the laboratory of Dr. Robert Lefkowitz.

Techniques: Binding Assay, Incubation, Expressing

Journal: Pharmacological research

Article Title: α 1 -adrenoceptor ligands inhibit chemokine receptor heteromerization partners of α 1B/D -adrenoceptors via interference with heteromer formation.

doi: 10.1016/j.phrs.2023.106730

Figure Lengend Snippet: Fig. 8. α1-AR ligands interfere with heteromerization between α1B/D-AR and their chemokine receptor partners. A. THP-1 cells were incubated with vehicle (ctrl., top), 10 μM phenylephrine (PE, center) or 10 μM phentolamine (PT, bottom) for 30 min at 37 ◦C and cell surface expression of receptor-receptor interactions visualized by PLA. Images show merged 4′,6-diamidino-2-phenylindole (DAPI, nuclear counterstain) and PLA signals (red, λexcitation/emission 598/634 nm) ac quired from z-stack images (n = 10; thickness 0.5 µm, bottom to top) and are representative of n = 3 independent experiments. Scale bar, 10 µm. B-G. Quantification of PLA signals for heteromers between α1B-AR and CCR1 (B), α1B-AR and CCR2 (C), α1B-AR and CXCR4 (D), α1D-AR and CCR1 (E), α1D-AR and CCR2 (F) and α1D-AR and CXCR4 (G) from n = 3 experiments. Data are mean ± SE, n = 3. * : p < 0.05 vs. ctrl.

Article Snippet: FLAG-CCR2-Tango was generated by insertion of CCR2 cDNA in the empty TANGO vector. α1b-AR and CCR2 cDNA were fused at Age I and Xba I sites with either RlucII or EYFP at C-terminus to form α1b-AR-RlucII and CCR2-EYFP, as described [14]. β-arrestin-2 mYFP was from Addgene (#36917) deposited by the laboratory of Dr. Robert Lefkowitz.

Techniques: Incubation, Expressing

Journal: Pharmacological research

Article Title: α 1 -adrenoceptor ligands inhibit chemokine receptor heteromerization partners of α 1B/D -adrenoceptors via interference with heteromer formation.

doi: 10.1016/j.phrs.2023.106730

Figure Lengend Snippet: Fig. 9. α1-AR ligands inhibit chemotaxis of leukocytes mediated by chemokine receptor heteromerization partners of α1B/D-ARs in vivo. A. Human CCL2 activates mouse and human CCR2. HEK293T cells were transfected with Gαi1-Rluc8, Gβ3, and Gγ9-GFP2 together with human or mouse CCR2. Forty-eight hours after transfection, cells were stimulated with human CCL2 and BRET measured. BRET change: BRET in the presence minus BRET in the absence human CCL2. Data are mean ± SE, n = 3. B-D. Analysis of leukocyte infiltration into dorsal air pouches of C57BL/6 mice. Mice received injections of vehicle (-) or 2.5 mg/kg LPS (B), 6 µg CCL2 (C), or 2 µg CXCL12 (D) plus vehicle or various doses of phenylephrine and phentolamine, as indicated, into the air pouches. 24 h later, air pouch cells were har vested and analyzed by flow cytometry. Leu kocytes (% ctrl.): Number of CD45 positive cells/mL in % of the number of cells/mL after vehicle injection (-). Data are mean ± SE, n = 3–4 mice/condition. * : p < 0.05 vs. injec tion of the chemotactic agent plus vehicle (+).

Article Snippet: FLAG-CCR2-Tango was generated by insertion of CCR2 cDNA in the empty TANGO vector. α1b-AR and CCR2 cDNA were fused at Age I and Xba I sites with either RlucII or EYFP at C-terminus to form α1b-AR-RlucII and CCR2-EYFP, as described [14]. β-arrestin-2 mYFP was from Addgene (#36917) deposited by the laboratory of Dr. Robert Lefkowitz.

Techniques: Chemotaxis Assay, In Vivo, Transfection, Flow Cytometry, Injection

Journal: Journal of Medicinal Chemistry

Article Title: Design and Characterization of an Intracellular Covalent Ligand for CC Chemokine Receptor 2

doi: 10.1021/acs.jmedchem.0c01137

Figure Lengend Snippet: Chemical structures and binding mode of sulfonamide derivatives. (a) Chemical structures of the parent compound SD-24, non-covalent control 7 , and the sulfonamide scaffold used for the design of putative covalent ligands. (b) Binding mode of compound 7 in the intracellular binding pocket of CCR2. (c) Docking of compound 7 into the crystal structure of CCR2 (PDB 5T1A); compound 7 makes a hydrogen bond with Cys75 ICL1 .

Article Snippet: Tango CCR2- bla osteosarcoma cells stably expressing human CCR2b (U2OS-CCR2) were obtained from Invitrogen (Carlsbad, CA).

Techniques: Binding Assay

Journal: Journal of Medicinal Chemistry

Article Title: Design and Characterization of an Intracellular Covalent Ligand for CC Chemokine Receptor 2

doi: 10.1021/acs.jmedchem.0c01137

Figure Lengend Snippet: Radioligand binding assays with preincubation and washing suggest a covalent interaction. (a, b) Time-dependent characterization of affinity of compounds 7 (a) and 14 (b). Concentration-dependent inhibition of [ 3 H]-CCR2-RA-[ R ] binding to U2OS-CCR2 cell membrane preparations following a 20 min coincubation which was preceded (4 h) or not (0 h) by preincubation with unlabeled ligands. Preincubation shifts the apparent affinity of compound 14 . pK i values obtained from non-linear regression fits of these data are described in Table . (c) [ 3 H]-CCR2-RA-[ R ] binding to U2OS-CCR2 membranes pretreated or not with iodoacetamide (IA) followed by preincubation with 10×IC 50 concentration of compounds 7 and 14 and repeated washouts. Following the washouts, the inhibition of radioligand binding by compound 14 persists in non-IA-treated membranes but is recovered in membranes pretreated with IA, indicating the involvement of cysteine residues. Data represent the mean ± SEM of at least three independent experiments performed in duplicate. Significant differences between indicated groups were determined using an unpaired t- test with Welch’s correction: ** p < 0.01, *** p < 0.001 or ns, not significant.

Article Snippet: Tango CCR2- bla osteosarcoma cells stably expressing human CCR2b (U2OS-CCR2) were obtained from Invitrogen (Carlsbad, CA).

Techniques: Binding Assay, Concentration Assay, Inhibition

Journal: Journal of Medicinal Chemistry

Article Title: Design and Characterization of an Intracellular Covalent Ligand for CC Chemokine Receptor 2

doi: 10.1021/acs.jmedchem.0c01137

Figure Lengend Snippet: Cysteine 75 seems to be involved in a covalent bond with compound 14 . (a) Docking of compound 14 into the crystal structure of CCR2 (PDB 5T1A), showing the cysteine residues with potential to interact with this ligand: Cys75 ICL1 within 4 Å; Cys70 ICL1 at 6.4 Å; and Cys232 ICL3 at 14 Å. (b) Proposed binding mode of compound 14 , displaying the covalent interaction between the thiocyanate warhead and Cys75 ICL1 . (c) Washout-radioligand experiments performed after preincubation of compound 14 (60 or 160 nM for triple mutant) or compound 7 (200 nM) in membranes from CHO cells transiently transfected with CCR2 mutants. Data represent the mean ± SEM of at least three independent experiments performed in duplicate. **** p < 0.0001, one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: Tango CCR2- bla osteosarcoma cells stably expressing human CCR2b (U2OS-CCR2) were obtained from Invitrogen (Carlsbad, CA).

Techniques: Binding Assay, Mutagenesis, Transfection

Journal: Journal of Medicinal Chemistry

Article Title: Design and Characterization of an Intracellular Covalent Ligand for CC Chemokine Receptor 2

doi: 10.1021/acs.jmedchem.0c01137

Figure Lengend Snippet: Binding Affinities (pK i ) of Synthesized Sulfonamide Derivatives Determined in [ 3 H]-CCR2-RA Displacement Assays b

Article Snippet: Tango CCR2- bla osteosarcoma cells stably expressing human CCR2b (U2OS-CCR2) were obtained from Invitrogen (Carlsbad, CA).

Techniques: Binding Assay, Synthesized

Journal: Journal of Medicinal Chemistry

Article Title: Design and Characterization of an Intracellular Covalent Ligand for CC Chemokine Receptor 2

doi: 10.1021/acs.jmedchem.0c01137

Figure Lengend Snippet: Time-Dependent Characterization of the Affinity (pK i ) of Compounds 7 and 14 Obtained from [ 3 H]-CCR2-RA-[ R ] Displacement Assays with U2OS-CCR2 Membranes d

Article Snippet: Tango CCR2- bla osteosarcoma cells stably expressing human CCR2b (U2OS-CCR2) were obtained from Invitrogen (Carlsbad, CA).

Techniques:

Journal: Journal of Medicinal Chemistry

Article Title: Design and Characterization of an Intracellular Covalent Ligand for CC Chemokine Receptor 2

doi: 10.1021/acs.jmedchem.0c01137

Figure Lengend Snippet: Binding Affinities (pIC 50 ) of 7 and 14 to Membranes of CHO Cells Expressing the Different CCR2 Constructs, Obtained from [ 3 H]CCR2-RA-[ R ] Displacement Assays a

Article Snippet: Tango CCR2- bla osteosarcoma cells stably expressing human CCR2b (U2OS-CCR2) were obtained from Invitrogen (Carlsbad, CA).

Techniques: Binding Assay, Expressing, Construct

Journal: Oncogene

Article Title: Induction of anti-VEGF therapy resistance by upregulated expression of microseminoprotein (MSMP).

doi: 10.1038/onc.2017.348

Figure Lengend Snippet: Figure 3. Secreted MSMP has a proangiogenic effect on endothelial cells. (a) Effects of medium conditioned with ovarian or lung cancer cells in which MSMP expression was or was not depleted by siRNA on tube formation by RF24 cells with or without the addition of rMSMP. Left: representative images of tube formation assays, magnification, × 20. Right: numbers of tubes in each assay in representative high-power fields. A two-tailed unpaired Student's t-test was used for statistical analysis. Data are shown as means ± s.d. of triplicate assays. CM, conditioned medium (scale bar, 100 µM). (b) Direct effects of rMSMP on RF24 tube formation with no or low concentrations of fetal bovine serum with growth factors. Left: representative images of tube formation assays. Right: numbers of tubes in each assay in representative high-power fields. A two-tailed unpaired Student's t-test was used for statistical analysis. Data are shown as means ± s.d. of triplicate assays (scale bar, 100 µM). (c) Effects of CCR2 silencing on RF24 tube formation with or without added rMSMP. Left: representative images of tube formation assays, magnification, × 100. Right: numbers of tubes in each assay in representative high-power fields. A two-tailed unpaired Student's t-test was used for statistical analysis. Data are shown as means ± s.d. of triplicate assays (scale bar, 100 µM). (d) Effects of CCR2 expression on RF24 tube formation. A two-tailed unpaired Student's t-test was used for statistical analysis. Data are shown as means ± s.d. of triplicate assays. (e) Left: immunoblot of phosphorylated ERK (pERK), ERK and B-actin expression in RF24 cells in which CCR2 was or was not silenced with or without the addition of rMSMP. Si-Control, control siRNA; si-CCR2, CCR2 siRNA. Right: effects of GSK1120212 on RF24 tube formation. A two-tailed unpaired Student's t-test was used for statistical analysis. Data are shown as means ± s.d. of triplicate assays.

Article Snippet: CCR2 expression plasmid was obtained from Addgene (CCR2-Tango).

Techniques: Expressing, Two Tailed Test, Western Blot, Control