Skip to main content
. Author manuscript; available in PMC: 2011 May 28.
Published in final edited form as: Immunity. 2010 May 6;32(5):654–669. doi: 10.1016/j.immuni.2010.04.011

Figure 1. Loss of LC3-II in HIV-1-Infected DCs and MyDCs Is Due to a Block in the Initiation Step of Autophagic Flux.

Figure 1

(A) Confocal immunofluorescence analysis of LC3 (red) in unexposed iDC (NI) or 20 hr HIV-1-exposed iDCs (HIV-1). DAPI (nucleus) is shown in gray. Quantification of absolute number of LC3+ puncta (lower graph). Data are means of 30 acquired fields (±SD) and representative of three experiments. Scale bars represent 5 μm.

(B) Confocal immunofluorescence of HIVGag (green) and LC3 (red) in unexposed or HIV-1 pulsed MyDCs for 20 hr. Data are representative of two independent experiments. Scale bars represent 5 μm.

(C) The left panel shows immunoblot analysis of LC3 in lysates of DC exposed to HIV-1 for indicated times. The right panel shows quantification of LC3-I and LC3-II levels normalized to actin. Actin expression at time 0 was considered 100%. Data are means (±SD) of four independent experiments.

(D) Graph representing quantification of LC3-II normalized to actin in DCs (see representative experiment in Figure S1). Error bars represent the mean ± SD of three independent experiments.

(E) Lysates of iDCs, preincubated or not for 2 hr with rapamycin (50 μg/ml) and/or 30 min with chloroquine (10 μM) when indicated and then left untreated or treated with HIV-1 for 12 hr were immunoblotted with anti-LC3 (upper panels). Arrows indicate migration of LC3-I and LC3-II forms. Equal sample loading was controlled with anti-actin (lower panels). Data are representative of three experiments.

(F) Immunoblot analysis of p62 in lysates of DCs nonexposed (NI) or pulsed with HIV-1 for indicated times. Experiments were done in two donors.

(G) Immunoblot analysis of p62 in lysates of MyDC nonexposed or pulsed with HIV-1 for indicated times. Experiments were done in two donors.

(H) Confocal analysis of DC pulsed or not pulsed with HIV-1 for 20 hr. Cells were fixed, permeabilized, and stained with anti-HIV-1Gag (green), anti-poly-Ub (red), and anti-Lamp1 (blue). Quantification of poly-Ub fluorescence signal intensity was measured in unexposed DCs (NI) or DCs pulsed with increasing doses of HIV-1 (50–500 ng p24). Data are means of fluorescence intensity (±SD) in 30 cells and representative of two experiments. Scale bars represent 5 μm.