The mevalonate pathway is a druggable target for vaccine adjuvant discovery. Yun xia et al
Cytosol, membrane and total cell Rab5 levels in BMDCs pre-treated with simvastatin. Immunofluorescence staining assay showing the co-localization of Lysotracker (red) and Alexa488-Dextran (green) in BMDCs
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BMDCs were plated at low density (less than 40% confluence) on sterile cover slips and pre-treated with simvastatin (1 μM) or Rab5 siRNA for 24 h. Alexa488-Dextran (Invitrogen) (100 μg/mL) and Lysotracker Red DND-26 (Life Technologies) (1 μM) were then added to cells for incubation (10 min) at 37 °C. Cells were then washed and fixed in 2% PFA (Twbio) in PBS for 15 min at 37 °C, followed by permeabilization buffer (Biolegend) for 5 min at 37 °C. Cells were mounted with DAPI-Fluoromount-G (SouthernBiotech) and imaging carried out by using a confocal microscope (Zeiss 780). Mice were sacrificed 5 days after influenza A (PR8) challenge, and lungs were perfused and fixed with 4% paraformaldehyde (PFA). Fixed lungs were embedded in paraffin and cut into 8 μm sections. Sections were stained with hematoxylin and eosin (H&E), then read under double-blind conditions.