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. 2019 Feb 12;15(2):e1007495. doi: 10.1371/journal.ppat.1007495

Fig 1. Ngo infection induces autophagic flux.

Fig 1

(A)Representative immunoblot showing LC3-I, LC3-II and GAPDH in ME180 cells that were mock infected, starved (St.) for 4 h, treated with EGFR inhibitor AG1478, or infected with Ngo strain MS11 at an MOI of 10 for 2, 4, 6 or 8 h. GAPDH served as the internal control for each sample. (B)Densitometry quantification of LC3-II levels in 5 independent immunoblots (described in A). LC3-II levels in each lane were normalized to the internal GAPDH signal, and the normalized values were expressed relative to that in mock-infected cells. Error bars represent standard error of the mean (SEM). Statistical analysis was performed using student’s t-test. (C)Representative immunoblot showing LC3-I, LC3-II and GAPDH in primary human endocervical epithelial cells that were mock infected, starved (St.) for 5 minutes, or infected with Ngo at an MOI of 10 for 2, 4, 6, or 8 h. GAPDH served as the internal control for each sample. (D)Densitometry quantification of LC3-II levels in 3 independent immunoblots (described in C). LC3-II levels in each lane were normalized to the internal GAPDH signal, and the normalized values were expressed relative to of that in mock-infected cells. Error bars represent standard error of the mean (SEM). Statistical analysis was performed using student’s t-test. (E)Representative deconvolution images of ME180 cells either mock or Ngo infected for 2, 4, or 6 h. Samples were stained with DAPI (blue) and LC3 antibody (red). (F)Quantification of LC3-positive puncta in ME180 cells treated as described in C, from 3 independent experiments. In each experiment, >25 images per condition were analyzed. The mid z-section of each image was analyzed for the number of LC3+ puncta using ImageJ software Analyze Particle function. Error bars represent SEM. Statistical analysis was performed using student’s t-test (G)Representative immunoblot (n = 2) showing LC3-I, LC3-II and GAPDH in ME180 cells that were treated with 0, 5, 10, or 20 μM of CQ for 30 min, then mock infected, starved (St.), or infected with Ngo at an MOI of 10.