fig4

Figure 4. APS-Lat-EXO reactivates HIV-1 provirus through the PI3K/AKT/NF-κB signaling pathway. (A) Representative fluorescence microscopy images showing GFP expression across different treatment conditions (20×). Cells were exposed to LY294002 (5 μM), MK2206 2HCl (0.5 μM), or JSH-23 (1 μM) as pathway inhibitors; (B, D, F and H) Immunoblot analysis of PI3K, phosphorylated PI3K, AKT, phosphorylated AKT, NF-κB, and phosphorylated NF-κB following 48 h exposure to APS-Lat-EXO, either alone or together with the indicated inhibitors; (C, E, G and I) Densitometric analysis corresponding to the immunoblot results; (J) Quantification of HIV-1 p24 levels in culture supernatants by ELISA (100× dilution); (K and L) RT-qPCR measurement of Gag and LTR transcript abundance in both supernatants and cellular fractions. Results are derived from at least three independent experiments, with individual data points representing separate samples. Statistical differences were determined using Tukey’s multiple comparisons test (C/E, n = 4; G, n = 5; I/J/K/L, n = 3). Data are presented as mean ± SD. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001; ^****P < 0.0001. APS: Astragalus polysaccharide; EXO: exosome; Lat-EXO: latency-reversing exosome; APS-Lat-EXO: APS-modified latency-reversing exosome; PI3K: phosphoinositide 3-kinase; AKT: protein kinase B; NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells; GFP: green fluorescent protein; LY294002: PI3K inhibitor; MK2206 2HCl: AKT inhibitor; JSH-23: NF-κB inhibitor; p24: HIV-1 capsid protein p24; ELISA: enzyme-linked immunosorbent assay; RT-qPCR: reverse transcription quantitative polymerase chain reaction; SD: standard deviation; Gag: group-specific antigen; LTR: long terminal repeat.