Fig 2: Exposure of microglia to cART resulted in blockade of autophagosome–lysosome fusion. (A) rPMs were seeded into a 12-well plate followed by tandem fluorescent-tagged MAP1LC3B plasmid. Next, cells were exposed to cART (5 µM each of TDF, FTC, and DTG) for an additional 24 h and observed by confocal imaging. The results showed that cART exposure significantly increased the formation of autophagosomes (yellow puncta). (B) Representative bar graph showing the number of autophagosome (yellow puncta) per cell. (C) Representative bar graph showing the number of autolysosome (red puncta) per cell. (D) rPMs were seeded into 12-well plates followed with cART exposure for 24 h. Cells were then double immunostained with MAP1LC3B and LAMP2 antibody and observed by immunofluorescent microscopy. (E,F) Representative bar graphs showing cART-mediated decreased LAMP2 puncta and decreased colocalization of MAP1LC3B and LAMP2. BAF—autophagosome fusion inhibitor, and rapamycin (RAP—autophagy inducer) were used as controls for autophagy flux. Data is from three independent experiments and is expressed as means ± SEM and were analyzed using one-way ANOVA. *, p < 0.05 vs. control.

Fig 3: Characterization of dendritic cell-derived EVs: Expression levels of dicer (A) and Lamp2b (B) in mouse DC2.4 dendritic cell line co-transfected with dicer siRNA and RVG-Lamp2b plasmid. (C) Extracellular vesicles (EVs) were isolated from the culture supernatants of engineered DC2.4 mouse dendritic cells by a series of centrifugation. (D) Zeta view count of EVs. (E) Size distribution of EVs. (F) Western blot images for the expression of CD63 and Lamp2 in purified EVs. All experiments were done at least three independent times, and representative figures are shown. Actin served as a loading control for western blots in B. *p < 0.05 vs. control.

Fig 4: Short-term SF had no impact on autophagy in the frontal cortex. (A,B) Beclin1, LC3II, and p62 did not change their levels in the striatum of SF-experienced mouse brains compared to the sham controls (* p > 0.05, n = 5). (C,D) LAMP1, LAMP2, and TFEB did not change their levels in the striatum of SF-experienced mouse brains compared to the Scheme 0. n = 5). For all Western blots, ß-actin were served as a protein load control. Data were expressed as means ± SEM and were analyzed using Student’s t-tests (* p < 0.05 versus sham).). (Original Western Blot Figure see Supplementary Figure S3).

Fig 5: HSPA overexpression abrogated cART-mediated impairment of lysosomal function. Control rPMs and heat shock protein family A (HSPA) overexpressing rPMs were seeded into six-well plates subjected to various treatments for 24 h. Protein homogenates were prepared for the detection of the indicated molecules. (A,B) Representative western blots showing overexpressing HSPA in rPMs reversed cART-mediated downregulation of LAMP2 and mCTSD expression levels. (C) Representative bar graph showing overexpression of HSPA in rPMs protected lysosomal pH. (D,E) Representative bar graphs showing HSPA protected LMP (D), and CTSD activity (E) in cART-treated rPMs. For all western blots, ACTB served as a protein loading control. Data is from three independent experiments and is expressed as means ± SEM and were analyzed using student t-test or one-way ANOVA. *, p < 0.05 vs. control; #, p < 0.05 vs. cART.