Figure 5.
Figure 5. PA exoproducts are sufficient to induce apoptotic lung epithelial cell death, in part through a secreted T2SS protein. Single-plane confocal images showing Mpl−/− mouse lung tissue at 0 hours (A) and 6 hours (B) after IT PA SN. Tissue was labeled with 4′,6-diamidino-2-phenylindole (nuclei, blue), TUNEL (red), phalloidin (actin, white), and anti-pro-SPC (type 2 cells, green). There was an increase in the number of TUNEL-positive cells at 6 hours, the majority of which were also positive for pro-SPC (indicated by white arrows). Magenta arrows indicate TUNEL-positive cells likely to be endothelial cells based on separate staining for CD31 as well as the shape and size of the nucleus. Scale bars represent 100 μm. (C) Kinetics of cell death as measured by a lactate dehydrogenase assay in MLE cells incubated with vehicle or PA SN. (D) Live-cell imaging showing cellular blebbing and annexin V staining (green) followed by propidium iodide staining (red). Scale bars represent 10 μm. (E) Fold change in MLE cytotoxicity 16 hours following the addition of 10% PA SN, where cells were pretreated with vehicle (dimethyl sulfoxide), pan-caspase inhibitor (Z-VAD-fmk), or RIP1-inhibitor (Nec-1s). Each point represents the median cytotoxicity of 3 to 4 technical replicates from a single trial relative to PA SN and vehicle treatment, which is set at 1.0. (F) Flow cytometry of MLE cells positive for a fluorogenic caspase-3/7 substrate 6 hours after the addition of 10% PA SN. (G) Cleaved caspase-3, total caspase-3, and β-actin protein expression in MLE cells after the addition of 10% PA SN at the indicated time points. Staurosporine served as a positive control for caspase cleavage in panels F and G. (H) Fold change in MLE cytotoxicity after the addition of PA SN or equal parts PA SN denatured for 30 minutes at 100°C. (I) Fold change in MLE cytotoxicity after the addition of 50% PA SN from the PA14 parent strain, PA14ΔxcpQ, or PA14ΔexoTUY. Each point represents median cytotoxicity from a single trial as described above. Comparison by Kruskal-Wallis test with Dunn’s test for multiple comparisons (E; the line represents post hoc analysis) or Mann-Whitney U test (I). *P < .05.

PA exoproducts are sufficient to induce apoptotic lung epithelial cell death, in part through a secreted T2SS protein. Single-plane confocal images showing Mpl−/− mouse lung tissue at 0 hours (A) and 6 hours (B) after IT PA SN. Tissue was labeled with 4′,6-diamidino-2-phenylindole (nuclei, blue), TUNEL (red), phalloidin (actin, white), and anti-pro-SPC (type 2 cells, green). There was an increase in the number of TUNEL-positive cells at 6 hours, the majority of which were also positive for pro-SPC (indicated by white arrows). Magenta arrows indicate TUNEL-positive cells likely to be endothelial cells based on separate staining for CD31 as well as the shape and size of the nucleus. Scale bars represent 100 μm. (C) Kinetics of cell death as measured by a lactate dehydrogenase assay in MLE cells incubated with vehicle or PA SN. (D) Live-cell imaging showing cellular blebbing and annexin V staining (green) followed by propidium iodide staining (red). Scale bars represent 10 μm. (E) Fold change in MLE cytotoxicity 16 hours following the addition of 10% PA SN, where cells were pretreated with vehicle (dimethyl sulfoxide), pan-caspase inhibitor (Z-VAD-fmk), or RIP1-inhibitor (Nec-1s). Each point represents the median cytotoxicity of 3 to 4 technical replicates from a single trial relative to PA SN and vehicle treatment, which is set at 1.0. (F) Flow cytometry of MLE cells positive for a fluorogenic caspase-3/7 substrate 6 hours after the addition of 10% PA SN. (G) Cleaved caspase-3, total caspase-3, and β-actin protein expression in MLE cells after the addition of 10% PA SN at the indicated time points. Staurosporine served as a positive control for caspase cleavage in panels F and G. (H) Fold change in MLE cytotoxicity after the addition of PA SN or equal parts PA SN denatured for 30 minutes at 100°C. (I) Fold change in MLE cytotoxicity after the addition of 50% PA SN from the PA14 parent strain, PA14ΔxcpQ, or PA14ΔexoTUY. Each point represents median cytotoxicity from a single trial as described above. Comparison by Kruskal-Wallis test with Dunn’s test for multiple comparisons (E; the line represents post hoc analysis) or Mann-Whitney U test (I). *P < .05.

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