Supplementary MaterialsS1 Fig: Cystic fibrosis isolates of aggregate in extruded apoptotic cells. adheres to useless over live cells. UV produced apoptotic wtMDCK cells were mixed with trypsin-detached Lifeact-GFP MDCK cells, stained with Annexin V-Alexa 647 and added to glass-grown wtMDCK monolayers followed by PAK-mCherry contamination and incubation for 3h. Projected confocal Z stack shows that PAK (red) preferentially adheres to lifeless cells (blue) over living cells (green). Scale bar 20 m.(PDF) ppat.1006068.s004.pdf (1.0M) GUID:?BC7F97E4-C1B4-4130-977A-AF11A7F876A0 S5 Fig: Efferocytosis takes place in cultured MDCK monolayers. Lifeact-GFP MDCK monolayers (green) were stained with Annexin V-Alexa 647 (blue) and incubated for 3 h. Confocal xy plane (top) and orthogonal section (bottom) showing an efferocytic phagosome. Scale bar: 5 m.(PDF) ppat.1006068.s005.pdf (1.8M) GUID:?E72E9224-D6E8-47F4-9EFE-682315717FD0 S6 Fig: Internalized cystic fibrosis isolates are inside cells that also have intracellular apoptotic cell debris. (A) Extruded apoptotic cells in transwell-grown MDCK monolayers were labeled with fluorescent Annexin V (green). Monolayers were then infected with the cystic fibrosis isolates. Strain 2b is usually shown (red). Epithelial cells are visualized by Phalloidin staining (blue). Scale bar: 10 m. (B) Percentage of internalized bacteria in cells that also have intracellular apoptotic cell debris.(PDF) ppat.1006068.s006.pdf (4.1M) GUID:?B94B3E2E-25BE-45C1-8B19-AA1C04F9E5C7 S7 Fig: internalizes into 16HBE14o- cells Rabbit Polyclonal to MRPS24 through efferocytosis. 16HBE14o- layers were stained with Annexin V-Alexa 488 (green), infected with PAK-mCherry (red) and incubated for 3 h. Samples had been set and stained with phalloidin for F-actin (blue). Confocal xy airplane (best) and orthogonal section (bottom level) displaying an intracellular vesicle formulated with both apoptotic cell particles and bacterias. Scale club: 5 m.(PDF) ppat.1006068.s007.pdf (1.8M) GUID:?7BF43CB1-D09D-44EA-8EB1-E3EB33D0B6BD S8 Fig: Consultant image showing the way the Object counter-top tool from ImageJ can be used to evaluate the quantity of monolayer-associated apoptotic cell materials. (A) CellTrace (blue) tagged apoptotic cells linked to lifeact-GFP monolayers (green). (B) Object or particle map rendered by the thing counter-top Nemorubicin tool. (C) Graph listing the quantity (in voxels) from the contaminants. The localization (i.e. extracellular or intracellular) of apoptotic materials was defined aesthetically.(PDF) ppat.1006068.s008.pdf (1.8M) GUID:?7D49B429-A24C-4A58-9A16-794B3EBFEF99 S9 Fig: Total monolayer-associated bacteria after pre-incubation with AnnexinV. Percentage of total monolayer-associated after pre-incubating transwell-grown lifeact-GFP MDCK monolayers with unlabeled Annexin V for 15 min in binding buffer or with binding buffer by itself (control). Data had been normalized to regulate. NS: not really significant.(PDF) ppat.1006068.s009.pdf (239K) GUID:?B4B1C426-734D-4240-B270-4A2895C637EC S10 Fig: Internalized apoptotic materials is certainly localized into LAMP1 vesicles. Transwell-grown MDCK monolayers had been stained with Annexin V-Alexa 647 (blue), contaminated Nemorubicin either with wtPAK (A) or PAK-GFP (B) and incubated for 3 h. (A) XY airplane showing a Light fixture1-positive vesicle formulated with apoptotic materials. F-actin: red, Light fixture1: green. (B) XY airplane showing a Light fixture1-positive vesicle containing apoptotic materials and bacterias. PAK-GFP: green, Light fixture1: red. Size pubs: 5 m.(PDF) ppat.1006068.s010.pdf (2.2M) GUID:?0F93D8A5-1734-4459-81C4-7E230EC31C48 S11 Fig: Antibiotics treatment kills surface-aggregated bacterias. Live imaging of MDCK monolayers contaminated with PAK. Bacterial viability after contact with Amikacin plus Carbenicillin was examined by staining live bacterias with SYTO 9 (green) and counterstaining useless bacterias with propidium iodide (reddish colored).(PDF) ppat.1006068.s011.pdf (2.5M) GUID:?52618A19-EC1A-484E-A47F-C0DEFEA25A50 S12 Fig: Epithelial cell viability. (A) Viability of MDCK cells through the entire intracellular PAK success curve was assayed by trypan blue exclusion (B) Annexin V staining was completed at 3, 6 and 9 h after infections of MDCK cells with PAK-GFP (antibiotics had been added 2 h after infections as referred to above). Cells had been stained with phalloidin. Amount of cells with or without intracellular bacterias and with or without apical Annexin staining was quantified. A Chi square check indicated that cells with internalized bacterias and cells with apical Annexin V staining are indie variables (3h: p = 0.54 NS, 6h p = 0.69 NS, 9h p = 0.83 NS).(PDF) ppat.1006068.s012.pdf (693K) GUID:?14AD9BFA-F681-4C82-9C9C-2AAAD1CB3378 S13 Fig: Nemorubicin Intracellular cystic fibrosis isolate 2b survival curve in MDCK cells. (PDF) ppat.1006068.s013.pdf (411K) GUID:?FD868AD1-296B-4844-BB8E-F251E54837C3 S14 Fig: inhabits LAMP1-positive vesicles inside 16HBE14o- cells. 16HBE14o- layers were infected with PAK for 3 h. Projected confocal Z stack (top) and orthogonal section (bottom) showing LAMP1-positive vesicles made up of bacteria. F-actin: blue, PAK-GFP: green and LAMP1: reddish. Scale.