Fig. 4.

Baf-A₁ at high concentration inhibits lysosome acidification and autophagosome flux in A549 cells. A: confocal imaging of lysosomes. Confocal micrographs showing LysoTracker Red DND-99 staining of A549 cells treated for 24 h with different concentrations of Baf-A₁ (0.1, 10, or 100 nM), prior to infection with A/PR/8/34 virus. Images were obtained 24 h after initiation of infection. Control cultures were not pretreated with Baf-A₁. B: histogram showing quantification of integrated fluorescence of LysoTracker Red DND-99 in A549 cells under conditions shown in A. By use of Olympus FluoView software, LysoTracker fluorescence was determined from 6 non-adjacent ×60 high-power fields with an average of 65 cells/field. Values are expressed as means ± SD compared with nontreated control cells of 2 independent experiments. ***P < 0.001 vs. bib-treated cells. C: epifluorescence micrographs showing labeling with pHRodoRed (red) in A549 cells treated with different concentrations of Baf-A₁ as indicated, included untreated (control) cultures. Cell nuclei were stained with Hoechst 333442 (blue). D: histogram showing integrated pHRodoRed Dextran (pRRD) fluorescence per cell in A549 cultures under conditions described for images in C. Fluorescence was determined from individual image fields by using Nikon NIS-Elements imaging software as described in materials and methods. Five distinct image areas were captured from individual wells. Mean background fluorescence was determined from 3 distinct areas devoid of cells and subtracted from pRRD fluorescence of each image field. The corrected fluorescence was normalized for cell number by dividing by total nuclear counts to obtain the total integrated pRRD fluorescence per cell (y-axis). For each well, mean corrected values per image field were averaged and used to determine mean ± SD from 3 independent experiments. ***P < 0.001 compared with untreated control cultures. E: immunoblot showing effects of Baf-A₁ on infection-induced LC3-II accumulation. A549 cells were pretreated with different concentrations of Baf-A₁ for 24 h prior to being infected with influenza A/PR/8/34 virus. The blot shown is typical of 3 independent experiments. As a control, infection burden was monitored by assessing NP accumulation. As a loading control GAPDH abundance was determined for each sample. F: densitometric analysis of LC3-II in A549 cells from immunoblots under conditions described for E. LC3-II levels were normalized to GAPDH as a loading control. ***P < 0.001 for uninfected control cells (C) vs. A/PR/8/34 virus-only infected cells (0). *P < 0.05 compared with A/PR/8/34 virus-only infected cells (0). #P < 0.01 compared to A/PR/8/34 virus-only infected cells (0). Results represent mean ± SD from 3 independent experiments.