Fig 2: Human breast cancer cells secrete CX3CL1 in an FGF-dependent manner to promote macrophage cell migration.FGF-dependent production and secretion of CX3CL1 by HS578T cells mediates recruitment of human macrophages. A) HS578T cells treated with 50ng/mL bFGF demonstrated significant induction of CX3CL1 gene expression after 4 hours relative to PBS solvent control-treated cells. B) Soluble CX3CL1 protein concentrations were significantly upregulated in HS578T cells treated with bFGF for 8 hours in comparison to control-treated cells. C) HS578T cells, which produce high levels of endogenous bFGF, were treated with the PD173074 for 8 hours to inhibit autocrine FGFR activation. A significant reduction in CX3CL1 gene expression was observed relative to DMSO-treated cells as measured by qRT-PCR. D) THP1 cells that had been differentiated into macrophages using PMA demonstrated increased migratory potential when exposed to conditioned medium from CX3CL1-expressing HS578T cells in comparison to serum free medium. Furthermore, this enhanced cell migration was significantly reduced when THP1 cells were treated with a CX3CL1 blocking antibody (aCX3CL1) relative to IgG-treated THP1 cells. *p<0.05, **p<0.001. Results in each figure panel are representative of a minimum of three different experiments. Error bars represent SEM.

Fig 3: CX3CR1 is required for keratinocytes differentiation. (A) Expression of CX3CR1 in parental PA-1 cell line and that stably transfected with either CX3CR1 or scrambled shRNA constructs was examined with Western blot. Actin served as a loading control. CX3CR1 expression levels were quantified using digital densitometry and normalized to the levels of actin expression. (B) Expression of cytokeratin 14 in PA-1 cells stably transfected with either CX3CR1 or scrambled shRNA constructs treated with recombinant BMP-4 or vehicle, as indicated, was examined with immunocytochemistry. CK14 in PA-1 was probed with anti-CK14 and Alexa555-conjugated anti-rabbit antibodies, and DNA was stained with 4’,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI); CK14 – red, DNA – blue. Images were taken using 5× magnification on the objective. The histogram demonstrates the average intensity of CK14 signal across the field as determined by the Zeiss AxioVision software. Data is an average of three independent experiments. *p < 0.05. (C) Expression of cytokeratins 14 and 18 in PA-1 cells stably transfected with either CX3CR1 or scrambled shRNA constructs treated with recombinant BMP-4 or vehicle, as indicated, was examined with Western blot. ß-Tubulin served as a loading control. The histogram shows CK14 and CK18 expression levels. Expression of CK18 in CX3CR1shRNA-transfected PA-1 cells was arbitrarily set as 1 and expression of both CK14 and CK18 in other conditions was calculated accordingly. The data represent a typical Western blot image, and quantitative analysis was performed using three independently performed experiments. *p < 0.05. (D) Expression of CX3CL1 in SKOV-3 and PA-1 cell lines was examined with Western blot. Actin served as a loading control. SKOV-3 cell line was used as a positive control.

Fig 4: Chemokine expression in type 1 diabetic (T1D) and healthy control (Control) pancreata. Pancreatic sections from healthy control subjects (case identification nos. 6117, 6112, and 6115) and type 1 diabetic donors (case identification nos. 6052 and 6087) were acquired through the nPOD program and stained for insulin, glucagon, and chemokines as detailed in RESEARCH DESIGN AND METHODS. Note the presence of some CCL5, CCL8, and CXCL9 in diabetic donors but their absence in healthy control samples. Only very faint CX3CL1 staining was observed in one of the type 1 diabetic samples (identification no. 6087). Scale bar: 20 µm. (A high-quality digital representation of this figure is available in the online issue.)

Fig 5: iFGFR1-induced CX3CL1 secretion by epithelial cells mediates macrophage migration.siRNA techniques were used to directly target CX3CL1 in order to determine the role of CX3CL1 in iFGFR1-mediated macrophage recruitment. A) CX3CL1 gene expression levels were significantly elevated in HC-11/R1 cells transfected with non-targeting (NT) siRNA. In comparison, CX3CL1 gene expression levels were significantly reduced in CX3CL1siRNA HC-11/R1 cells treated with B/B after 24 hours. B) ELISA was performed to verify reduction in CX3CL1 protein secretion in HC-11/R1 cells transfected with CX3CL1siRNA. Results indicated that soluble CX3CL1 protein concentrations were significantly induced in HC-11/R1 cells transfected with non-targeting (NT) siRNA. In comparison, soluble protein concentrations were reduced in CX3CL1siRNA HC-11/R1 cells treated with B/B after 24 hours. C) Conditioned medium from non-targeting HC-11/R1 cells treated with B/B significantly increased migration of RAW 264.7 cells relative to conditioned medium from cells treated with solvent alone. Exposure to conditioned medium from CX3CL1 siRNA HC-11/R1 cells significantly decreased RAW 264.7 cell migration. Furthermore, addition of 50ng/mL rmCX3CL1 to CX3CL1siRNA HC-11/R1 cells at the time of B/B treatment rescued the loss of RAW 264.7 cell migration in cells exposed to conditioned medium from CX3CL1siRNA HC-11/R1 cells. These results indicate that CX3CL1 is a key mediator of macrophage recruitment. *p<0.05, **p<0.001, ***p<0.0001. Results in each figure panel are representative of a minimum of three different experiments. Error bars represent SEM.