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. 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 6. RNA Interference or HIV-1-Mediated Autophagy Inhibition Dampen TLR4- and TLR8-Mediated Responses in DCs.

Figure 6

(A) Immunoblot analysis of LC3 and ATG5 expression in lysates from siCtrl, siLC3, and siATG5 DCs. Arrows denote LC3-I and LC3-II migrating forms.

(B) FACS analysis of TLR4-mediated intracellular TNF-α response upon 20 hr LPS stimulation of siCtrl, siLC3, or siATG5 DCs previously unexposed (NI) or pulsed with HIV-1 for 8 hr. BrefeldinA (10 μg/ml) was added 1 hr after adding LPS.

(C) TNF-α responses upon 20 hr TLR4 stimulation in non-transfected (NT), siCtrl, siLC3, and siATG5 DCs. Response was normalized to nonstimulated NT-DCs, and depicted as fold increase over control. Data represented means (±SD) of fold increase in TNF-α responses from five independent experiments.

(D) TNF-α responses in DCs exposed or not with HIV-1 for 8 hr and then stimulated with TLR8 agonist for 20 hr. Response was normalized to nonstimulated NT DCs and depicted as fold increase over control. Data represented means (±SD) of fold increase in TNF-α responses from five independent experiments.

(E) FACS analysis of TLR4-mediated intracellular TNF-α response upon 20 hr LPS stimulation of nontransfected (NT), siCtrl, or siLC3 DCs previously unexposed (NI) or pulsed with HIV-1 or rgp120 (5 mg/ml) for 8 hr. BrefeldinA (10 mg/ml) was added 1 hr after adding LPS. Intracellular TNF-α response was normalized to unstimulated NT DCs and represented on a graph (right) as fold increase over control. One representative experiment out of three is shown.

(F) DCs untreated (NI), or treated with HIV-1 or HIV-1-ΔEnv for 20 hr, were kept unstimulated or stimulated with LPS (1 μg/ml) for 15 min, in presence or absence of proteasome inhibitor Mg132 pretreatment when indicated (30 min at 1 μM). Lysates were then immunoblotted with anti-p176/180-IKKα/β, anti-IKK-α, anti-p32 IκB-α, anti-IκB-α, anti-p536 NF-κB, and anti-NF-κB. Sample loading was controlled with anti-actin. One representative experiment out of three is shown.

(G) The same experiment as in 6F was done on siCtrl, siATG5, and siLC3 DCs. One representative experiment out of three is shown.