Figure 4.
Morphine Astrocyte EV-Mediated Induction of lincRNA-Cox2 Involves TLR7 and NF-κB
(A) Mouse primary microglial cells were pre-treated with either TLR7 inhibitor (chloroquine 25 μM) or NF-κB inhibitor (SC-514, 5 μM) for 1 hr, followed by exposure of cells for 4 hr to EVs (2 μg of EVs per 2 × 105 cells) isolated from astrocyte conditioned media. Total RNA isolated from cells was analyzed by real-time PCR for expression of lincRNA-Cox2. (B) Mouse primary microglial cells were isolated from wild-type and TLR7 KO pups, and exposed to EVs isolated from morphine-stimulated astrocyte conditioned media for 4 hr. Total RNA from cells was analyzed by real-time PCR for expression of lincRNA-Cox2. (C) Representative confocal images of ISH assay using a probe specific for lincRNA-Cox2 (red) combined with DAPI staining (blue) and immunostaining for GAPDH (green) in mouse primary microglia. Scale bar, 10 μm. (D) Subcellular fractionation followed by qPCR for lincRNA-Cox2, U1 snRNA, and GAPDH mRNA. All values are shown as the mean ± SEM. *p < 0.05 versus control; #p < 0.05 versus treatment.