Figure 2. Intravenous injection of nanoparticle-encapsulated siRNA results in knock down in monocytes.
(a) PCR analysis of FACS-sorted splenic Ly-6Chigh monocytes after intravenous administration of nanoparticle-encapsulated siRNA targeting CCR2 (siCCR2). siCON: control siRNA treatment. n = 3 per group. (b) Western blot of Ly-6Chigh monocytes isolated from the spleen of mice that were either treated with siCCR2 or control. The reduced band in the siCCR2 lane confirms a lower expression of CCR2 receptor protein in splenic monocytes after intravenous treatment (experiment was done in triplicate, representative blot is cropped. The full length blot is presented in Supplementary Figure 6). (c) FACS analysis of CCR2 protein on splenic Ly-6Chigh monocytes. Representative histogram shows isotype antibody staining (black), control treatment (red) and siCCR2 treatment (blue). Bar graph of mean fluorescent intensity (MFI, y axis starts at mean fluorescence intensity of the isotype control; n = 6 per group). (d) Migration assay of sorted Ly-6Chigh monocytes using MCP-1 as chemoattractant. Cells were harvested from mice injected with either siCCR2 or control siRNA (n = 3 per group). Stained membranes on the left, bar graph shows enumeration of migrated cells. Mean ± SD, * P < 0.05.