Fig 2.

Transfection of poly(I · C) induces autophagy. (A) Immunoblot analysis of phosphorylated eIF2α (P-eIF2α) and PKR (P-PKR) proteins in HeLa cells. Cells were transfected with 0, 5, or 10 μg/ml poly(I · C) for 4 h. Total eIF2α and PKR blotting was used as a loading control. (B) Representative images of HeLa GFP-LC3 cells (scale bar, 10 μm) transfected for 4 h with poly(I · C) and quantification of GFP-LC3-positive cells with GFP-LC3 dots. As a control, HeLa cells were transfected with a GFP-LC3ΔG. The results are the means of three independent experiments, and 50 cells were analyzed per assay. *, P < 0.05; **, P < 0.01 (t test). (C) Immunoblot analysis of LC3-II protein in HeLa cells. Cells were transfected with poly(I · C) and either left untreated or treated with bafilomycin A1 (Baf) for 4 h. Actin blotting was used as a loading control. (D) HeLa cells were transfected with the mRFP-GFP-LC3 plasmid and treated with poly(I · C) for 4 h. Autolysosomes were quantified by counting the number of GFP⁻ RFP⁺ puncta per cell. The results are the means of three independent experiments. Twenty cells were analyzed per assay. *, P < 0.05 (t test). Amino-acid-starved cells were used as a positive control.