Fig. 2

Expression of Nef does not affect ATG9A localization, ATG9A levels or autophagy. a HeLa cells, control (−) or transiently expressing HIV-1 Nef-GFP (+), were fixed, permeabilized, immunostained with antibodies to endogenous ATG9A or TGN46, and imaged by confocal microscopy. Scale bar: 10 μm. Insets show enlarged views of the boxed areas. b, c Lysates of Jurkat or HeLa cells stably expressing GFP (−) or HIV-1 Nef-GFP (+) were subjected to SDS-PAGE and immunoblotting with antibodies to ATG9A, LC3 or actin. The positions of molecular mass markers (in kDa) are indicated on the left. ATG9A and LC3 levels were quantified relative to actin levels. Bar graphs represent the mean ± SD from four independent experiments. Values were normalized to the ATG9A/actin ratio in the absence of Nef. Statistical significance was determined using an unpaired Student’s t-test; ns: not significant (p > 0.05). d, e HeLa (d) or Jurkat (e) cells, stably expressing GFP (−Nef) or Nef-GFP (+Nef), were treated for different times with 10 μM chloroquine (CQ). Cell lysates were analyzed by SDS-PAGE and immunoblotting with antibodies to the indicated proteins. The positions of molecular mass markers (in kDa) are indicated on the left. Bar graphs represent the mean ± SD of the levels of LC3-I and LC3-II relative to the level of actin from three independent experiments. Values were normalized to the levels at time 0 in the absence of Nef. Statistical significance was analyzed using a one-way ANOVA followed by a Holm-Šídák post hoc test; ns: not significant (p > 0.05)