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: 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