Fig. 1

Rac1 siRNA efficiently inhibited CM-induced ramification. a Primary mouse microglia were transfected with Rac1 siRNAs or Scrambled siRNA (used as control) for 4 days, then incubated in normal medium or CM for 4 h and stained with the microglia surface marker isolectin B4 (green fluorescence). Microglia morphology was assessed on a fluorescence microscope. Scale bars, 20 μm. b High magnification of cells shown in a and the typical black and white threshold images of the cells generated by image analysis program Fiji/ImageJ are shown as an example. The outlines of each cell were drawn to measure cell perimeter and area, which are the parameters needed to calculate the circularity values (correspond to numbers near each outline). Note that cells touching the image edges were automatically excluded from the measurement. Scale bars, 20 μm. c Microglia were transfected with three individual siRNA oligonucleotides targeting Rac1 or a pool of them (3x siRNA Rac1) and then incubated with CM. Isolectin B4-stained microglia were visualized by microscopy, and the cell shape was quantified using the circularity value. Non-transfected cells (not tr) or cells transfected with Scrambled siRNA were used as controls of reference. Data are mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 vs. Scrambled siRNA