Figure 6.

Triggering through C40.T4 showed augmentation in autophagy. DCs infected with Mtb were stimulated through C40.T4, CD40, TLR-4 for 24 h and assessed for (A) NO in SNs by Greiss assay. (B) Expression of LC3 was detected in the whole cells lysates of DCs stimulated for 2 h through C40.T4 by immunoblotting. Actin was used as a loading control. Densitometric data show the LC3I/actin and LC3II/actin ratio. (C) LC3 puncta formation was demonstrated by immunofluorescence staining. Starved DCs were used as a positive control; (D,E) Bar graphs depict the percentage of LC3 puncta positive cells and LC3 puncta per cells, respectively. (F) DCs were stimulated through C40.T4 for 5 h were later incubated with acridine orange for 15 min to visualize autophagosomes by fluorescence microscopy (40×). Orange dots indicate the acidic vacuoles. (G) Beclin^KD DCs were infected with Mtb followed by stimulation with C40.T4. Later, cells were lysed and bacterial burden was quantified by CFU assay. Data represented as the mean ± SD (A,D,E,G); immunoblotting (B); fluorescent microscopy (C,F); are of 2–3 independent experiments. “*” and “**” indicate p < 0.05 and p < 0.01, respectively.