Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: CCR5 Inhibition Prevents Cardiac Dysfunction in the SIV/Macaque Model of HIV

doi: 10.1161/JAHA.114.000874

Figure Lengend Snippet: CCL5 decreases cardiomyocyte contractility via CCR5. Confocal microscopy showing expression of (A) CCR5 (red) on isolated adult rhesus macaque ventricular cardiomyocyte (RM VCM) merged with (B) sarcomeric actin (green). C, Representative twitch traces for VCM sequentially exposed to human recombinant CCL5 (100 nmol/L), then CCL5 (100 nmol/L) with maraviroc (MVC, 500 nmol/L). Compared to basal conditions (grey, left), CCL5 decreased contractility, shown by the reduced, flattened twitch amplitude (red, middle). The CCL5‐induced decline in contractility was reversed by subsequent addition of MVC with CCL5 (green, right). D, Representative twitch traces for reciprocal experiments showing VCM sequentially exposed to MVC (500 nmol/L) followed by a combination CCL5 (100 nmol/L) and MVC (500 nmol/L). Addition of MVC (blue, middle) did not alter contraction from basal (grey, left). Furthermore, addition of MVC prior to MVC+CCL5 (turquoise, left) prevented CCL5‐induced changes in contractility with twitch traces unchanged from basal. E, Summary data for RM VCM exposed to CCL5 (100 nmol/L) followed by MVC (500 nmol/L) with CCL5 (100 nmol/L). CCL5 significantly decreased fractional shortening compared to basal. Subsequent addition of MVC modulated the CCL5 effect towards basal shortening. F, CCL5 significantly increases the time from basal to 50% peak sarcomere length (t to pk) compared to basal conditions. Subsequent CCL5+MVC modulated t to pk shortening towards basal conditions. G, CCL5 did not significantly change the time from peak shortening to 50% baseline (t to bl) compared to basal conditions although t to bl in cells treated with CCL5+MVC was shorter than both basal and CCL5 treatment. Paired t ‐tests, mean value indicated by the top of bar with bars representing standard error.

Article Snippet: Cells were sequentially exposed to recombinant human CCL5 (278‐RN, R&D Systems) 100 nmol/L alone and with 500 nmol/L of MVC (NIH/AIDS Reagent).

Techniques: Confocal Microscopy, Expressing, Isolation, Recombinant

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) 3×10 5 THP-1 macrophages were treated with 15 mM lactate for 24 h, and the mRNA levels of chemokines were measured by quantitative PCR. The growth medium of control macrophages was titrated to pH6.1 using sterile HCl. (B) 3×10 5 THP-1 macrophages were incubated with different concentrations of lactate for 24 h, and CCL5 gene expression was determined with quantitative PCR. (C) 10 6 THP-1 macrophages were exposed to increasing concentrations of lactate for 48 h, and the secretion of CCL5 was measured by ELISA. (D) 10 6 human primary macrophages from breast cancer patients (n=9) were cultured with different concentrations of lactate for 48 h, and CCL5 production was detected. (E) 10 6 MDA-MB-231 cells were pre-treated with 15μM GSK 2837808A for 2 h, then the media were changed, and cells were cultured for another 24 h. The conditional media (MD-231 CM) were collected and applied to 10 6 THP-1 macrophages. CCL5 concentrations were detected with ELISA. (F) Immunohistochemical staining of CD68 and CCL5 in tumor adjacent tissues (control) and breast tumors (n=28). Scale bars represent 50 μm. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Real-time Polymerase Chain Reaction, Control, Sterility, Incubation, Gene Expression, Enzyme-linked Immunosorbent Assay, Cell Culture, Immunohistochemical staining, Staining

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) 10 6 THP-1 macrophages were treated with 15 mM lactate for 48 h, and the expression of key regulators in Notch, NF-κB, STAT3 and HIF, were detected by western blot. (B) 3×10 5 THP-1 macrophages were cultured with 15 mM lactate for 24 h, and the mRNA levels of Notch ligands and receptors were measured by quantitative PCR. (C) Western blot for Notch ligands and receptors in THP-1 (10 6 ) macrophages after 48 h lactate treatment. (D) Lactate stimulated the expression of NICD in a time and dose-dependent manner. 10 6 THP-1 macrophages were treated with 15 mM lactic acid for 48 h. Data presented were representatives of at least three independent experiments. (E) 10 6 THP-1 macrophages were transfected with 50nM siNotch1, or pretreated with 50μM DAPT for 2 h, and then cultured with 15 mM lactate for 48 h. The secretion of CCL5 was measured by ELISA. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Western Blot, Cell Culture, Real-time Polymerase Chain Reaction, Transfection, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) 10 6 THP-1 macrophages were treated with 15 mM lactate for 72 h, and then cells were washed twice and fresh media were added. Macrophages were cultured for another 24 h and the conditional media (lactate CM) was collected. The effect of CM on breast cancer cell migration was measured by double chamber transwell assay. 5μg/ml anti-CCL5 neutralizing antibody significantly decreased lactate CM-induced cell migration. (B) 10 6 MCF-7 cells were co-cultured with 15 mM lactate-activated macrophages in the presence of 5μg/ml anti-CCL5 antibody or not, and protein levels of EMT markers were tested by western blot. (C) 10 6 breast cancer cells were co-cultured with 10 6 lactate-activated THP-1 macrophages (or 10 6 lactate-activated primary macrophages) for different time points, and the expression of CCR5 was monitored by western blot. (D) MDA-MB-231 and MCF-7 cells were transfected with shCCR5 plasmids, or pre-treated with 5μM Maraviroc for 2 h, then cell migration induced by lactate CM was detected by double chamber transwell assay. Lactate CM was described in (A). (E) MCF-7 cells (10 6 ) were transfected with pcDNA3.1-CCR5, and then cultured with 10ng/ml CCL5 for 24 h. The expression of E-cadherin, N-cadherin and vimentin was investigated by western blot. (F) 10 6 Human primary macrophages (No. 4 and No. 9) were treated with 15 mM lactate for 72 h and CM was collected as described in (A). The migration of MDA-MB-231 cells was measured in the presence of primary macrophage CM. 5μg/ml anti-CCL5 neutralizing antibody, shRNAs designed against CCR5, or 5μM Maraviroc, significantly reduced primary macrophage CM-induced cell migration. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Migration, Transwell Assay, Western Blot, Expressing, Transfection

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) 3×10 5 MDA-MB-231 and MCF-7 cells were stimulated with 1-5ng/ml TGF-β1 for 24 h, and total RNA was isolated and tested for CCR5 mRNA by quantitative PCR. (B) Western blot for CCR5 protein in breast cancer cells (10 6 ) under TGF-β1 stimulation for 48 h. Data presented were representatives of at least three independent experiments. (C) MDA-MB-231 and MCF-7 cells (3×10 5 ) were co-transfected with pGL3-CCR5 and pRL-TK and exposed to different concentrations of TGF-β1 for 24 h, and luciferase activities were determined. (D) MDA-MB-231 and MCF-7 cells were pre-treated with 5μM SIS3 for 2 h, and cells were subjected to luciferase assay. (E) 10 6 MCF-7 cells were transfected with TGFβRI/ALK5 siRNA, and were then co-cultured with lactate-activated THP-1 macrophages (ratio 1:1) for 24 h. The protein levels of CCR5 were assayed by western blot. (F) The expression of TGF-β1, CCL5 and CCR5 in clinical samples obtained from breast cancer patients. The mRNA levels were measured by quantitative PCR, and the correlation between TGF-β1 and CCL5-CCR5 axis was shown. (G) Representative IHC staining for TGF-β1, CCL5 and CCR5 in breast cancer samples. The sample used was derived from 28 breast cancer cases. Scale bars represent 50 μm. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Isolation, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Luciferase, Cell Culture, Expressing, Immunohistochemistry, Derivative Assay

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) Glucose uptake, lactic acid production and ATP levels in breast cancer cells co-cultured with lactate-activated THP-1 macrophages, with or without 5μg/ml anti-CCL5 neutralizing antibody. The co-culture system was described in Figure . (B) Western blots for glycolytic enzymes in breast cancer cells treated as in (A). (C) MDA-MB-231 cells were transfected with shRNAs designed against CCR5, or pre-treated with 5μM Maraviroc for 2 h, and then subjected to cell co-culture. Glucose uptake, lactic acid production and ATP levels were measured after co-culture. The co-culture system was described in Figure . (D) The protein levels of HK2, PKM2 and LDHA in MDA-MB-231 cells cultured as in (C). (E) Recombinant human CCL5 induced aerobic glycolysis in breast cancer cells. MDA-MB-231 and MCF-7/CCR5 cells were treated with increasing concentrations of CCL5 for 12 h, and glucose uptake, lactic acid production and ATP levels were detected. (F) Western blots for glycolytic enzymes in MDA-MB-231 and MCF-7/CCR5 cells after stimulation with CCL5. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Co-Culture Assay, Western Blot, Transfection, Recombinant

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) Western blot for AMPK, c-Myc, HIF-1α and Akt in breast cancer cells co-cultured with 15mM lactic acid-activated THP-1 macrophages (ratio 1:1) for 72 h. Results presented were representatives of at least three independent experiments. (B) The expression of AMPK downstream signaling target ACC in breast cancer cells co-cultured as in (A). (C) MDA-MB-231 and MCF-7 cells were transfected with 50 nM AMPKα1 siRNA, or pretreated with 10μM compound C for 4 h, and then incubated with 15mM lactic acid-activated THP-1 macrophages (ratio 1:1) for 48 h. The glucose uptake, lactic acid production and ATP levels were detected. (D) The inhibition of AMPK abrogated macrophage-induced EMT in MCF-7 cells. Cells were treated as described in (C). After co-culture, the expression of EMT markers, E-cadherin and vimentin, was measured by western blot. (E) Recombinant human CCL5 induced the phosphorylation of AMPK in MDA-MB-231 and MCF-7/CCR5 cells. 10 6 cells were treated with 50ng/ml CCL5 for defferent time points as indicated, and phosphorylated AMPK and total AMPK were investigated by western blot. (F) Inhibition of CCR5 in MDA-MB-231 cells significantly attenuated macrophage-induced AMPK phosphorylation. MDA-MB-231 cells were transfected with shRNAs designed against CCR5, or pre-treated with 5μM Maraviroc for 2 h, then co-cultured with 15 mM lactate-activated macrophages as described in (A). After co-culture, the phosphorylation of AMPK was detected by western blot. (G) Expressions of CCL5, CCR5 and p-AMPK in samples obtained from breast cancer patients (n =28). Scale bars represent 50 μm. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Western Blot, Cell Culture, Expressing, Transfection, Incubation, Inhibition, Co-Culture Assay, Recombinant, Phospho-proteomics

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) MDA-MB-231 cells were co-cultured with 15 mM lactate-activated THP-1 macrophages for 7 days, in the presence of 5μg/ml anti-CCL5 neutralizing antibody or not. MDA-MB-231 cells were then collected and injected into the tail vein of nude mice. After two weeks, animals were sacrificed and metastatic nodules on lung surfaces were counted. (B) CCR5, HK2 and p-AMPK were immunostained in MDA-MB-231 metastases. Scale bars represent 50 μm. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Injection

Journal: PLoS ONE

Article Title: GEC-derived SFRP5 Inhibits Wnt5a-Induced Macrophage Chemotaxis and Activation

doi: 10.1371/journal.pone.0085058

Figure Lengend Snippet: (A) and (B) Real-time PCR and ELISA showed that CCL2 mRNA expression and protein secretion was upregulated by transfection with Wnt5a expression vector, * P <0.01. (C) and (D) Real-time PCR and ELISA showed that CCL2 mRNA expression and protein secretion was stimulated by rWnt5a (0.1 µg/ml, 0.5 µg/ml, 1 µg/ml, respectively), * P <0.01. (E) Immunofluorescence showed that both Wnt5a transfection and rWnt5a treatment increased CCL2 production. Control V, transfection with control vector; Wnt5a V, transfection with Wnt5a expression vector. Data are expressed as mean±SD, n = 3.

Article Snippet: Quantikine ELISA kits (Boster, Wuhan, China) were used to determine the concentrations of CCL2, CCL5, CCL7, TNF-α, IL-1β, IL-6 and PGE 2 in cell (2×10 5 ) culture supernatants according to the manufacturer’s instructions.

Techniques: Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Expressing, Transfection, Plasmid Preparation, Immunofluorescence, Control

Journal: PLoS ONE

Article Title: GEC-derived SFRP5 Inhibits Wnt5a-Induced Macrophage Chemotaxis and Activation

doi: 10.1371/journal.pone.0085058

Figure Lengend Snippet: (A) Western blot showed that both Wnt5a transfection and rWnt5a treatment (0.5 µg/ml) enhanced the expression of phosphorylated JNK (P-JNK). (B) and (C) Real-time PCR and ELISA showed that JNK inhibitor SP600125 (10 µmol/L) inhibited CCL2 induction by Wnt5a transfection or rWnt5a treatment (0.5 µg/ml). * P <0.01. (D) Western blot showed that both Wnt5a transfection and rWnt5a treatment (0.5 µg/ml) increased the expression of phosphorylated p65 (P-p65). (E) and (F) Real-time PCR and ELISA showed that NFκB inhibitor BAY 11-7082 (10 µmol/L) suppressed CCL2 induction by Wnt5a transfection or rWnt5a treatment (0.5 µg/ml). * P <0.01. Cont, control; ContV, control vector; WntV, Wnt5a vector; rWnt, rWnt5a; SP, SP600125; BAY, BAY 11-7082. Data are expressed as mean±SD, n = 3.

Article Snippet: Quantikine ELISA kits (Boster, Wuhan, China) were used to determine the concentrations of CCL2, CCL5, CCL7, TNF-α, IL-1β, IL-6 and PGE 2 in cell (2×10 5 ) culture supernatants according to the manufacturer’s instructions.

Techniques: Western Blot, Transfection, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Control, Plasmid Preparation

Journal: PLoS ONE

Article Title: GEC-derived SFRP5 Inhibits Wnt5a-Induced Macrophage Chemotaxis and Activation

doi: 10.1371/journal.pone.0085058

Figure Lengend Snippet: (A) Transwell assay showed that conditioned-medium from macrophages treated with Wnt5a expression vector promoted cell migration; the induction in cell migration was suppressed by CCL2 neutralizing antibody AF-479-NA (0.1 µg/ml). * P <0.01. (B) Transwell assay showed that conditioned-medium from macrophages treated with rWnt5a (0.5 µg/ml) increased cell migration; the increased cell migration was abrogated when CCL2 expression was silenced by CCL2 siRNA in macrophages. * P <0.01. (C) Transwell assay showed that conditioned-medium from macrophages treated with Wnt5a expression vector promoted cell invasion; the induction in cell invasion was suppressed by CCL2 neutralizing antibody AF-479-NA (0.1 µg/ml). * P <0.01. (D) Conditioned-medium from macrophages treated with Wnt5a expression vector induced cytoskeletal changes. Cont, control; ContV, control vector; WntV, Wnt5a vector; rWnt, rWnt5a; CCLsiR, CCL2 siRNA; ContsiR, control siRNA; AF, AF-479-NA. Data are expressed as mean±SD, n = 3.

Article Snippet: Quantikine ELISA kits (Boster, Wuhan, China) were used to determine the concentrations of CCL2, CCL5, CCL7, TNF-α, IL-1β, IL-6 and PGE 2 in cell (2×10 5 ) culture supernatants according to the manufacturer’s instructions.

Techniques: Transwell Assay, Expressing, Plasmid Preparation, Migration, Control

Journal: PLoS ONE

Article Title: GEC-derived SFRP5 Inhibits Wnt5a-Induced Macrophage Chemotaxis and Activation

doi: 10.1371/journal.pone.0085058

Figure Lengend Snippet: (A) and (B) Real-time PCR and ELISA showed that conditioned medium from SFRP5-positive GES-1 inhibited Wnt5a-induced CCL2 expression in macrophages; the inhibitory effect of the condition medium was abolished when SFRP5 expression was knocked down in GES-1. * P <0.01. (C) and (D) Real-time PCR and ELISA showed that conditioned medium from SFRP5-transfected BGC-803 suppressed Wnt5a-induced CCL2 expression in macrophages. * P <0.01. (E) and (F) Real-time PCR and ELISA showed that rSFRP5 inhibited CCL2 induction by Wnt5a transfection in a dose-dependent manner. * P <0.01. (G) Transwell assay showed that cell migration induced by conditioned medium from Wnt5a-transfected macrophages was suppressed by co-culture of Wnt5a-transfected macrophages with GES-1 or SFRP5-transfected BGC-803. * P <0.01. (H) Transwell assay showed that cell migration induced by conditioned medium from Wnt5a-transfected macrophages was inhibited by pretreatment of macrophages with rSFRP5. * P <0.01. Cont, control; ContV, control vector; WntV, Wnt5a vector; GES, GES-1; SFsiR, SFRP5 siRNA; ContsiR, control siRNA; BGC, BGC-803; SFV, SFRP5 vector; ContV, control vector. Data are expressed as mean±SD, n = 3.

Article Snippet: Quantikine ELISA kits (Boster, Wuhan, China) were used to determine the concentrations of CCL2, CCL5, CCL7, TNF-α, IL-1β, IL-6 and PGE 2 in cell (2×10 5 ) culture supernatants according to the manufacturer’s instructions.

Techniques: Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Expressing, Transfection, Transwell Assay, Migration, Co-Culture Assay, Control, Plasmid Preparation

Journal: PLoS ONE

Article Title: GEC-derived SFRP5 Inhibits Wnt5a-Induced Macrophage Chemotaxis and Activation

doi: 10.1371/journal.pone.0085058

Figure Lengend Snippet: (A–F) Real-time PCR and ELISA showed that mRNA expression and protein secretion of IL-1β, IL-6 and TNF-α were upregulated in macrophages by Wnt5a transfection; the upregulation of these cytokines was inhibited by JNK inhibitor SP600125 (10 µmol/L) and NFκB inhibitor BAY 11-7082 (10 µmol/L), respectively. * P <0.01. (G) and (H) Real-time PCR and ELISA showed that COX-2 expression and PGE 2 production were stimulated by Wnt5a transfection; the induction of COX-2 and PGE 2 was suppressed by BAY 11-7082 (10 µmol/L), but not by SP600125 (10 µmol/L). * P <0.01. Cont, control; ContV, control vector; WntV, Wnt5a vector; SP, SP600125; BAY, BAY 11-7082. Data are expressed as mean±SD, n = 3.

Article Snippet: Quantikine ELISA kits (Boster, Wuhan, China) were used to determine the concentrations of CCL2, CCL5, CCL7, TNF-α, IL-1β, IL-6 and PGE 2 in cell (2×10 5 ) culture supernatants according to the manufacturer’s instructions.

Techniques: Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Expressing, Transfection, Control, Plasmid Preparation

Journal: PLoS ONE

Article Title: GEC-derived SFRP5 Inhibits Wnt5a-Induced Macrophage Chemotaxis and Activation

doi: 10.1371/journal.pone.0085058

Figure Lengend Snippet: (A–H) Real-time PCR and ELISA showed that conditioned medium from SFRP5-positive GES-1 inhibited Wnt5a-induced IL-1β, IL-6, TNF-α and COX-2/PGE 2 expression in macrophages; the inhibitory effect of the condition medium was abolished when SFRP5 expression was knocked down in GES-1. * P <0.01. Cont, control; ContV, control vector; WntV, Wnt5a vector; GES, GES-1; SFsiR, SFRP5 siRNA; ContsiR, control siRNA. Data are expressed as mean±SD, n = 3.

Article Snippet: Quantikine ELISA kits (Boster, Wuhan, China) were used to determine the concentrations of CCL2, CCL5, CCL7, TNF-α, IL-1β, IL-6 and PGE 2 in cell (2×10 5 ) culture supernatants according to the manufacturer’s instructions.

Techniques: Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Expressing, Control, Plasmid Preparation

Journal: PLoS ONE

Article Title: GEC-derived SFRP5 Inhibits Wnt5a-Induced Macrophage Chemotaxis and Activation

doi: 10.1371/journal.pone.0085058

Figure Lengend Snippet: (A–H) Real-time PCR and ELISA showed that conditioned medium from SFRP5-transfected BGC-803 suppressed Wnt5a-induced IL-1β, IL-6, TNF-α and COX-2/PGE2 expression in macrophages. * P <0.01. Cont, control; ContV, control vector; WntV, Wnt5a vector; BGC, BGC-803; SFV, SFRP5 vector; ContV, control vector. Data are expressed as mean±SD, n = 3.

Article Snippet: Quantikine ELISA kits (Boster, Wuhan, China) were used to determine the concentrations of CCL2, CCL5, CCL7, TNF-α, IL-1β, IL-6 and PGE 2 in cell (2×10 5 ) culture supernatants according to the manufacturer’s instructions.

Techniques: Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Transfection, Expressing, Control, Plasmid Preparation

Journal: PLoS ONE

Article Title: GEC-derived SFRP5 Inhibits Wnt5a-Induced Macrophage Chemotaxis and Activation

doi: 10.1371/journal.pone.0085058

Figure Lengend Snippet: Wnt5a chemoattracts macrophages by upregulate CCL2 expression, and activates macrophages to secrete cytokines, which are blocked by GEC-derived SFRP5.

Article Snippet: Quantikine ELISA kits (Boster, Wuhan, China) were used to determine the concentrations of CCL2, CCL5, CCL7, TNF-α, IL-1β, IL-6 and PGE 2 in cell (2×10 5 ) culture supernatants according to the manufacturer’s instructions.

Techniques: Expressing, Derivative Assay