Fig. 2

Autophagy targets HIV Gag for degradation. A HEK293T cells were transfected with HIV-1 NL4-3 Δnef proviral DNA and treated with rapamycin (4 μM), chloroquine (60 μM) and/or ALLN (25 μM) for 12 h. 48 h later, lysates were analyzed by western blot for gp120, SQSTM1, p55, ACTB, and LC3. Densitometric analyses were performed to determine the relative ratios of gp120, SQSTM1 and p55. B HEK293T cells were co-transfected with Gag-EGFP, EGFP-LC3B or an empty vector. 48 h later, cells were harvested, and Gag was immunoprecipitated. The pulldown fraction was examined for SQSTM1 and LC3. Lysates were also analyzed by western blot for SQSTM1, Gag, LC3 and ACTB. C HEK293T cells were transfected with the HIV-1 NL4-3 provirus or an empty retroviral vector. 48 h later, cells were harvested, and LC3 was immunoprecipitated. The pulldown fraction was examined for LC3, SQSTM1, p55, gp120 and Nef. Lysates were also analyzed by western blot for gp120, SQSTM1, p55, Nef, LC3 and ACTB. D HEK293T cells treated with an irrelevant siRNA (si-ctr) or SQSTM1-specific siRNAs were transfected with HIV-1 NL4-3 Δnef proviral DNA. 48 h post-transfection, cells were harvested and endogenous LC3 was immunoprecipitated. The pulldown fraction was examined for LC3 and p55. Lysates were analyzed by western blot for SQSTM1, p55, LC3 and ACTB. E HEK293T cells were co-transfected with EGFP-LC3B and HIV-1 NL4-3 Δnef proviral DNA. Cells were exposed for 4 h to rapamycin (4 μM) or DMSO prior to microscopy visualization for EGFP-LC3 (green), Gag (red) and the nuclei (blue). Scale bar: 10 μm. All images are representative of three independent experiments