Figure 3
Figure 3. P17 chemokine–like activity is mediated specifically by the IL-8 receptor CXCR1. (A) Static adhesion of monocytes on fibrinogen. Monocytes were stimulated for 2 minutes at 37°C with PBS (NT) or GST or pretreated for 1 hour at 37°C with 50 μg/mL of mAb to CXCR1, CXCR2, or CXCR4 or with an unrelated mAb (Ctrl mAb) and then stimulated for 2 minutes with p17 (1μM). Bars represent the means ± SD of 3 independent experiments performed in triplicate. Statistical analysis was performed by paired 2-tail Student t test. ***P < .001. (B) Transwell migration assay of monocytes in response to the indicated treatments. Monocytes were stimulated for 90 minutes at 37°C with PBS (NT) or GST or pretreated for 1 hour at 37°C with 50 μg/mL of mAb to CXCR1, CXCR2, or CXCR4 or with Ctrl mAb and then stimulated with p17 (50nM). Bars represent the means ± SD of 3 independent experiments performed in duplicate. Statistical analysis was performed by paired 2-tail Student t test. ***P < .001. (C) Analysis of CXCR1 gene expression performed using quantitative real-time PCR. Monocytes were nucleoporated with scr siRNAs used as a negative control or with a pool of 4 distinct siRNAs specific for 4 distinct regions of CXCR1. Analysis of real-time PCR data were performed with the 2−ΔΔCt method using relative quantitation study software. Quantification of CXCR1 mRNA was normalized in each reaction according to the internal β-actin control. Bars represent the means ± SD of 3 independent experiments performed in triplicate. (D) Effect of CXCR1 silencing on surface-receptor expression. Monocytes nucleofected with scr siRNA or CXCR1 siRNA were incubated with isotype control Ab (solid histogram) or mAb to CXCR1 (open histogram), and then stained with APC-conjugated secondary Ab and analyzed by flow cytometry. (E) Transwell migration assay of monocytes nucleoporated with CXCR1 siRNAs or with scr siRNAs in response to the indicated treatments. Bars represent the means ± SD of 3 independent experiments performed in duplicate. Statistical analysis was performed by paired 2-tail Student t test. ***P < .001.

P17 chemokine–like activity is mediated specifically by the IL-8 receptor CXCR1. (A) Static adhesion of monocytes on fibrinogen. Monocytes were stimulated for 2 minutes at 37°C with PBS (NT) or GST or pretreated for 1 hour at 37°C with 50 μg/mL of mAb to CXCR1, CXCR2, or CXCR4 or with an unrelated mAb (Ctrl mAb) and then stimulated for 2 minutes with p17 (1μM). Bars represent the means ± SD of 3 independent experiments performed in triplicate. Statistical analysis was performed by paired 2-tail Student t test. ***P < .001. (B) Transwell migration assay of monocytes in response to the indicated treatments. Monocytes were stimulated for 90 minutes at 37°C with PBS (NT) or GST or pretreated for 1 hour at 37°C with 50 μg/mL of mAb to CXCR1, CXCR2, or CXCR4 or with Ctrl mAb and then stimulated with p17 (50nM). Bars represent the means ± SD of 3 independent experiments performed in duplicate. Statistical analysis was performed by paired 2-tail Student t test. ***P < .001. (C) Analysis of CXCR1 gene expression performed using quantitative real-time PCR. Monocytes were nucleoporated with scr siRNAs used as a negative control or with a pool of 4 distinct siRNAs specific for 4 distinct regions of CXCR1. Analysis of real-time PCR data were performed with the 2−ΔΔCt method using relative quantitation study software. Quantification of CXCR1 mRNA was normalized in each reaction according to the internal β-actin control. Bars represent the means ± SD of 3 independent experiments performed in triplicate. (D) Effect of CXCR1 silencing on surface-receptor expression. Monocytes nucleofected with scr siRNA or CXCR1 siRNA were incubated with isotype control Ab (solid histogram) or mAb to CXCR1 (open histogram), and then stained with APC-conjugated secondary Ab and analyzed by flow cytometry. (E) Transwell migration assay of monocytes nucleoporated with CXCR1 siRNAs or with scr siRNAs in response to the indicated treatments. Bars represent the means ± SD of 3 independent experiments performed in duplicate. Statistical analysis was performed by paired 2-tail Student t test. ***P < .001.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal