Journal: International Journal of Molecular Sciences

Article Title: The Autophagy Nucleation Factor ATG9 Forms Nanoclusters with the HIV-1 Receptor DC-SIGN and Regulates Early Antiviral Autophagy in Human Dendritic Cells

doi: 10.3390/ijms24109008

Figure Lengend Snippet: DC-SIGN engagement induces autophagy and associates with LC3. ( A ) HEK293T cells were co-transfected with plasmids expressing DC-SIGN WT and GST-expressing constructs as indicated. Cells were engaged or not with DC-SIGN mAbs (5 μg/mL) for 30 min before pull-down. Lysates (input control) and GST pull-down were immunoblotted with anti-DC-SIGN ( upper panels), anti-GST ( middle panels), and anti-GAPDH for input control. This experiment is representative of 4. ( B ) Lysates from MoDC treated for indicated times with DC-SIGN specific mAbs were immunoblotted with anti-LC3, and loading was controlled with anti-GAPDH ( upper blots). Negative control experiments were performed using lysates of cells incubated with non-specific mouse serum (Isotype Ab) and immunoblotted as before ( lower blots). Pre-treatments of MoDC with chloroquine (50 μM) were performed to validate functional autophagy flux as evidenced by LC3-II increase. Densitometry values and LC3-II/GAPDH ratio obtained from lysates of primary MoDC treated with DC-SIGN mAbs (n = 6) or Isotype mouse Abs (n = 3) were obtained for each time point of each condition and graphically reported on the right. The same experiment as above was performed but with lysates from MoDC treated with ( C ) ManLAM (2 μg/mL) (n = 4) or ( D ) challenged with HIV-1-R5 (MOI of 2) (n = 3) for indicated times. LC3-II/GAPDH ratio from densitometry analyses was graphically represented as before. Statistical significance: * = p < 0.05; ** = p < 0.01.

Article Snippet: Chemiluminescence was acquired (Chemidoc, Bio-Rad Laboratories, Hercules, CA, USA), followed by densitometry analysis (Image Lab TM software; Bio-Rad Laboratories, Hercules, CA, USA).

Techniques: Transfection, Expressing, Construct, Control, Negative Control, Incubation, Functional Assay

Journal: International Journal of Molecular Sciences

Article Title: The Autophagy Nucleation Factor ATG9 Forms Nanoclusters with the HIV-1 Receptor DC-SIGN and Regulates Early Antiviral Autophagy in Human Dendritic Cells

doi: 10.3390/ijms24109008

Figure Lengend Snippet: ATG9 is required for DC-SIGN-mediated autophagy flux activation. Primary MoDC were treated with irrelevant siRNA (siCtrl) or siRNA against ATG9 (siATG9), and ATG9 expression was controlled by RT-qPCR ( left graph). Lysates from MoDC transfected as above and pre-treated with bafilomycin A1 (50 nM) for 1 h before stimulation with ManLAM (2 μg/mL) for 2 h were immunoblotted with anti-LC3 ( upper blot). The loading control was performed with anti-actin ( lower blot). LC3-II/actin ratio from densitometry analyses obtained from 3 independent experiments (n = 3) was normalized to untreated controls of each siRNA condition and graphically represented ( right graph). Statistical significance: * = p < 0.05.

Article Snippet: Chemiluminescence was acquired (Chemidoc, Bio-Rad Laboratories, Hercules, CA, USA), followed by densitometry analysis (Image Lab TM software; Bio-Rad Laboratories, Hercules, CA, USA).

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Transfection, Control