Figure 4.

CCL7 Is the Functional Target of miR-23b

(A) Matched sequence of CCL7 3′ UTR-binding site. (B) Draining lymph nodes were collected from EAE mice at day 10 p.i. (C) CNS was collected at day 17 p.i. Tissues were submitted to RNA extraction and cDNA production. Analysis of expression of genes of interest was carried out by RT-PCR. (D) EAE mice treated with LV-miR23b or LV-Ctrl were sacrificed at day 17 p.i. Brain and spinal cord tissues were harvested and homogenized for western blot. (E) Densitometry was determined by ImageJ and data were analyzed by Graphpad Prism. (F) Schematic of bi-directional fluorescence reporter vector. (G) HEK293 cells were transfected with pDual-GFP/mCherry-CCL7 3′ UTR 3 days before transfection with miR-23b mimic or control mimic, as described in the Materials and Methods. After 24 hr, cells were checked under fluorescence microscopy. (H) Statistical analysis of (G) by Image-Pro Plus software. (I) Sorting GFP-positive cells by FACSAria. Flow cytometry analysis of GFP⁺ cell population before (upper level) and post-sorting (lower level) is shown. (J) CCL7 expression level was performed by qPCR. Data are mean ± SD (n = 5 each group) and analyzed by unpaired Student’s t test (**p < 0.01, ***p < 0.001, and ****p < 0.0001).