HIV-1 Tat, gp120, and CQ de-acidified endolysosomes. (A,B) Treatment of cells with CQ (30 µM), Tat (100 nM), and gp120 (1 nM) significantly (p < 0.0001) de-acidified endolysosome pH. Endolysosome de-acidification was shown in real time (arrowhead indicates the timepoint when each compound was added) (A) and as peak responses within 30 min (B). (C) Within 30 min, CQ (30 µM), Tat (100 nM), and gp120 (1 nM) significantly (p < 0.0001) decreased the H+ concentration of endolysosomes compared to controls. (D) At 24 h, CQ (30 µM), Tat (100 nM), and gp120 (1 nM) significantly (p < 0.0001) de-acidified endolysosome pH and (E) decreased [H+]. A one-way ANOVA with a Tukey’s post hoc multiple comparisons test was used for analysis. Each data point represents the mean pH measurement of 50 endolysosomes from five cells performed independently five times for each group (n = 250). Cells were chosen randomly in the microscope’s field of view with 25 cells used for experimentation per group; no cells were intentionally excluded. **** p < 0.0001.