HERV-K Env was endocytosed via CD98 receptors in human cortical neurons (HCNs). A, Representative spinning-disk confocal microscope images of HCNs labeled for Alexa Fluor 488 microtubule-associated protein 2 (MAP2). B, Representative spinning-disk confocal microscope images of CD98 receptors on the surface of HCNs stained with CD98 mAb and Alexa Fluor 488 sary antibody (green) and Hoechst 33342-positive nuclei (blue). C, Representative spinning-disk confocal microscope images of HCNs stained for HERV-K Env labeled with a recombinant Alexa Fluor 647 anti-6X His-tag antibody (red), LysoTracker-positive endolysosomes (green), and Hoechst 33342-positive nuclei (blue). HCNs were incubated with buffer (CTL), a CD98 monoclonal antibody (CD98 mAb, 30 min preincubation, 0.2 µg) and HERV-K Env (30 nM) for 1 h. Qualitatively, HERV-K Env colocalized with LysoTracker-positive endolysosomes (Merged, yellow) and this was blocked by CD98 mAb. The insets demonstrate a single LysoTracker-positive endolysosome (green) containing HERV-K Env (red). D, Quantitatively, HERV-K Env was significantly colocalized with LysoTracker-positive endolysosomes; Pearson's correlation coefficient was 0.50. This colocalization was blocked by CD98 mAb. An ANOVA with a Tukey's post hoc multiple-comparisons test was used for analysis. Mean ± SD as well as individual data points were included on each bar. Each data point represented the mean fluorescence of 300 endolysosomes from ten cells performed independently from three cell-culture preparations for each group (n = 9,000). n = total number of endolysosomes for each group plotted. Cells were chosen randomly in the microscope's field of view with 30 cells used for experimentation per group and no cells were intentionally excluded. Scale bar = 20 µm. D, F_(3,16) = 59.46, p < 0.0001.