Figure 1: HIV-1 pseudovirions (HIVpps) trafficked from early to late endosomes and into LC3+ compartments.
WT mouse platelets (1 × 109/mL) were incubated with Gag-mCherry HIVpps at 37°C for 5 to 120 min and prepared for immunofluorescence as described in the Methods. Platelets were stained with (A-B) anti-Rab4 antibody, (C-D) anti-Rab7 antibody, and then with Alexa 680-conjugated secondary antibodies, after permeabilization (see Methods). The Alexa 680-Rab signal was faux-colored blue to improve contrast. Images were taken using the 3D-SIM module of Nikon Ti-E N-STORM/N-SIM super-resolution microscope. Scale bar is 1 μm. Pearson’s correlation coefficient was calculated using the NIS-Elements v3.2 N-SIM/STORM suite software to show overlap between: (B) Alexa 680-Rab4 and Gag-mCherry HIVpps, (D) Alexa 680-Rab7 and Gag-mCherry HIVpps, at the indicated time points. Data are representative of 2 independent experiments. (E-F) Platelets (1 × 109/mL) from GFP-LC3 transgenic mice were incubated with Gag-mCherry HIVpps at 37°C for 5 to 120 min and imaged and analyzed as above. Data are representative of 3 independent experiments.