Journal: American Journal of Cancer Research

Article Title: Oncolytic Newcastle disease virus induces autophagy-dependent immunogenic cell death in lung cancer cells

doi:

Figure Lengend Snippet: Effects of depletion of autophagy-related genes on NDV-triggered ICD determinants. (A) Confirmation of the depletion efficiency in A549 cells stably depleted of ATG5 (shATG5) or LC3 (shLC3) by immunoblot analysis. β-actin was used as a loading control. ATG5- or LC3-depleted A549 cells (B) and H1650 cells (C) were infected or mock-infected with NDV/FMW (MOI = 1) for 24 in A549 cells and 24 and 36 h in H1650 cells. Flow-cytometric analysis of CRT exposure was performed (Figure 2A). Data are shown as Mean ± S.D. for three independent experiments (*P<0.05, n.s = not significant). (D) ATG5- or LC3-depleted A549 cells and control cells were infected or mock-infected with NDV/FMW (MOI = 1) for 36 h. Confocal analysis of CRT exposure was carried out (Figure 2B). MTX was used as a positive control, arrowheads indicate positive area. Representative images are shown for three independent experiments. (E) IB analysis of released HMGB1 in whole cell lysates and concentrated supernatants of NDV/FMW-infected A549, ATG5- and LC3-depleted A549 cells. Blots shown are representative of two independent experiments. (F) A549 cells were treated with vehicle, Rapamycin (1 μM) or BEZ235 (1 μM), then they were infected with vehicle or NDV/FMW (1 MOI) for 24 h. Flow-cytometric analysis for CRT exposure. Representative images are shown for three independent experiments.

Article Snippet: The following lentiviral constructs were purchased from Santa Cruz: ATG5 shRNA (#sc-41445-V), MAP LC3β shRNA (#sc-43390-V) and noncoding shRNA (#sc-108080).

Techniques: Stable Transfection, Western Blot, Control, Infection, Positive Control