Effect of PolyI:C-stimulated hCMEC/D3 culture SN on the activation of ISGF3, STAT3, and MAPK in macrophages. (A) hCMEC/D3 cells were stimulated with 0.1, 1, and 2.5 µg/mL PolyI:C for 16 hours, then washed 3 times, and further incubated in fresh medium to 48 hours poststimulation. SN was collected to treat macrophages for 6 hours (10% v/v). (B) Macrophages were treated with 10% (v/v) of SN from 1 µg/mL PolyI:C-stimulated hCMEC/D3 culture for the indicated time periods. (C) Macrophages were treated with indicated percentage (v/v) of SN from 1 µg/mL PolyI:C-stimulated hCMEC/D3 culture for 6 hours. Protein was extracted and western blot was performed to examine the expression of ISGF-3γp48. Representative blots from 3 independent experiments ae shown. Densitometry analysis of the blot was performed by using ImageJ 1.44 software (National Institutes of Health) and plotted into graphs using data collected from triplicate experiments. Asterisks indicate that the differences between the SN-treated and control cells are statistically significant (*P < .05; **P < .01). (D) Macrophages were treated with 10% (v/v) of SN from indicated concentrations of PolyI:C stimulated hCMEC/D3 cultures for 6 hours. (E) Macrophages were treated with 10% (v/v) of SN from 1 μg/ml PolyI:C-stimulated hCMEC/D3 culture for indicated time points or with indicated percentage (v/v) of SN from 1 μg/ml PolyI:C stimulated hCMEC/D3 cutlure for 6 hours. Protein was extracted after treatment and western blot was performed to examine the expression of STAT3, p-STAT3, p-JNK, p-Erk1/2 amd GAPDH. Representative data from three times independent experiments was shown. Vertical lines have been inserted to indicate repositioned blot lanes.