Figure 5.

MVs inhibit autophagy induced by purified LLO. HEK293-GFP-LC3 cells were incubated with 200 μg MVs (isolated from EGDe bacterial cultures grown for 40 h) for 4.5 h before autophagy was induced with 250 ng of purified LLO for 1.5 h. Confocal microscopic images show (A) mock-treated cells (PBS), (B) cells treated with only LLO and (C) cells treated with MVs + LLO. (D) is a magnified area of the white square in (B), and (E) is magnified area of the white square in (C). Scale bar: 10 μm. Green dots represent GFP-LC3 puncta (arrows). (F) HEK293-GFP-LC3 cells were incubated with MVs and LLO in the same way as for confocal microscopy. Host cell lysates were analyzed for GFP-LC3 lipidation by immunoblot using monoclonal GFP antiserum (upper panel). The membrane was reprobed using anti-α-actin antibody as an internal control. The ratio of GFP-LC3-II to α-actin is presented in arbitrary units (lower panel). Immunoblots show (a) mock-treated cells, (b) cells treated with only LLO, and (c) cells treated with MVs + LLO. Data was quantified, normalized and shown from three independent experiments. ^**P < 0.01, Student's t-test.