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. 2024 May 27;27(6):110131. doi: 10.1016/j.isci.2024.110131

Figure 3.

Figure 3

KOs alter virus entry and the expression of HIV-1 Gag and autophagy proteins LAMP1 and LC3B-II

(A and B) WT and PICALM KO cell lines were infected with WT NL4-3 or -heat-inactivated virus containing BlaM-Vpr (MOI = 1) for 2 h and virus fusion was evaluated by the expression of cleaved CCF2 (acquired in Pacific blue channel, LSR Fortessa BD biosciences). (A) Representative dot plots and (B) bar graphs of % of cells + for cleaved CCF2 (BlaM +).

(C and D) WT and PICALM KO cells were infected with NL4.3 for 48 h and CD4-BV421 expression were analyzed by Flow cytometry.

(E) Data representative of uninfected WT and PICALM KO stained for CXCR4 BB700. WT and PICALM KO were infected with NL4.3 for 48 h (MOI 5, n = 3), and 2 h before harvesting, treated with BafA1 (100 nM), then collected for IF, coverslips were treated with 0.1% Saponin to deplete free cytoplasmic LC3BI, then stained for Gag (AF488), LAMP1 (AF647), LC3B (AF594) and DAPI.

(F and G) Representative images and plotted data showing (H) LAMP1, (I) LC3B-II expression, and (J) LAMP1 and LC3B-II colocalization. Relative values (%) were calculated by setting WT values to 100%. μm, micrometer; KO, knock out; ∩, colocalization (intersection); UI, uninfected; Inf., infected. Ո, colocalization (i.e., intersection). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; 1-way ANOVA, Tukey post-test.