Figure 7.

Dynamin-dependent endocytosis is likely involved in MV-mediated induction of IFN-β in RAW 264.7 cells. (A) RAW 264.7 cells were either treated with 80 µM Dynasore, which inhibits dynamin-mediated endocytosis, or with PBS buffer for 30 min and incubated with bMVs (5 µg/mL total protein) for 3 h, homogenized, and processed for qPCR analysis of β-actin and IFN-β mRNA expression. (B) Schematic representation of endosomal TLR signaling inhibition by the endosomal acidification inhibitors bafilomycin and chloroquine. Acidification of the endosome is essential for TLR3, TLR7, and TLR9 signaling in response to nucleic acid binding. Inhibition of the endosomal acidification process by bafilomycin A1 or chloroquine treatment prevents proteolytic cleavage of endosomal TLRs thereby inhibiting their signaling activity. (C) RAW 264.7 cells were either treated with PBS, 0.5 µM bafilomycin A1, and/or 100 µM chloroquine for 30 min followed by stimulation with bMVs (5 µg/mL) for 3 h, homogenized, and analyzed by qPCR for β-actin and IFN-β mRNA expression. (D) Association of MV RNA with an early endosomal marker. Macrophages were incubated for 10 min with SYTO RNASelect-stained MVs (1 µg/mL), washed, fixed, and immunostained with anti-EEA1 antibodies. Arrows indicate “moderate colocalization” between MV-RNA and EEA1.