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. 2014 May;88(10):5706–5717. doi: 10.1128/JVI.03142-13

FIG 2.

FIG 2

SIV MA-induced monocyte migration is mediated specifically by CXCR1. (A) Transwell migration assay of monocytes in response to PBS (NT), GST, p17, or SIV MA. Cells were stimulated for 90 min at 37°C with proteins at a concentration of 1 μg/ml. Bars represent the means ± the SD of three independent experiments performed in duplicate. Statistical analysis was performed by one-way ANOVA, and a Bonferroni's post test was used to compare data (***, P < 0.001). (B) Transwell migration assay of monocytes in response to the indicated treatments. Monocytes were stimulated for 90 min at 37°C with PBS (NT) or pretreated for 1 h at 37°C with 50 μg of MAb to CXCR1, MAb to CXCR2, or Ctrl MAb/ml and then stimulated for 90 min at 37°C with SIV MA (1 μg/ml). Bars represent the means ± the SD of three independent experiments performed in duplicate. Statistical analysis was performed by one-way ANOVA, and a Bonferroni's post test was used to compare data (***, P < 0.001). (C) Analysis of CXCR1 gene expression performed by using quantitative real-time PCR. Monocytes were nucleoporated with scr siRNAs used as a negative control or with SMARTpool siRNAs specific for four distinct regions of CXCR1. Analysis of real-time PCR data was performed using the 2−ΔΔCT method and relative quantitation study software. Quantification of CXCR1 mRNA was normalized in each reaction according to the internal β-actin control. Bars represent the means ± the SD of three 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 antibody (solid histogram) or MAb to CXCR1 (open histogram), stained with APC-conjugated secondary antibody, 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 ± the SD of three independent experiments performed in duplicate. Statistical analysis was performed by paired two-tailed Student t test (***, P < 0.001). (F) CXCR1 expression in Jurkat cells is sufficient to mediate chemotaxis in response to SIV MA. Transwell migration assay of Jurkat cells transfected with pEGFP-N3 or pEGFP-N3-CXCR1 in response to the indicated treatments. Bars represent the means ± the SD of three independent experiments performed in duplicate. Statistical analysis was performed by one-way ANOVA, and a Bonferroni's post test was used to compare data (**, P < 0.01; ***, P < 0.001).