To rule out the possibility that formation of the structure was simply delayed, we performed EM at later time points after contamination. and HMEC-1 cells were starved in EBSS medium, and collected samples were stained with anti-LC3 antibody at the indicated time points. Images were acquired by confocal Pedunculoside microscopy. Scale bar, 10 m. Formation of LC3 puncta is usually depicted by the bar graph. Data represent the means SD from three impartial experiments. (G) Cells were treated with 10% FBS complete medium or EBSS medium with or without bafilomycin A1 (100 nM) for 2 h, and then subjected to detect protein levels of LC3 and GAPDH by western blot analysis. The data show that there was Rabbit Polyclonal to PKCB no difference in autophagic flux between two cell types.(TIF) ppat.1006444.s001.tif (1.5M) GUID:?98CF0178-D3F9-46EF-A3C5-26DF72E80F63 S2 Fig: GAS infection induces LC3 puncta formation and lipidation, but not formation of double-membrane structure surrounding GAS in endothelial cells. (A) HMEC-1 cells were infected with GAS at MOI = 1, 5, 10, and 25, or heat-killed GAS at MOI = 25, for 2 h. (B) Cells were infected with GAS at MOI = 25 and collected at the indicated time points post-infection. Gentamicin was added to kill extracellular bacteria 30 min after contamination. Samples were collected for western blot analysis to detect LC3-I/II conversion. (C) GFP-LC3Cexpressing HMEC-1 cells were infected with GAS at MOI = 5 for various times and then observed by fluorescence microscopy. The proportion of cells with GFP-LC3 puncta is usually shown as a percentage of total GFP-expressing and GAS-infected HMEC-1 cells. Scale bar, 10 m. (D) HMEC-1 cells were infected with GAS for 1 h, and then treated with gentamicin to kill extracellular bacteria. Cells were collected at the indicated time points post-infection and fixed for electron microscopy. White arrowheads indicate GAS within vesicles at early stages, and black arrows indicate GAS in the cytoplasm in late stage. No isolation membrane was detected at any time point post-infection. GAS division occurs at all stages post-infection. Scale bar, 5 m Pedunculoside for upper and 1 m for below.(TIF) ppat.1006444.s002.tif (3.1M) GUID:?3861D19D-2D36-43AA-8764-2C77ADEFD139 S3 Fig: LC3 and Gal3-positive GAS is not surrounded by double membrane structure in endothelial cells. (A-D) Representative images of correlative light electron microscopy of GAS-infected cells. GFP-LC3 and Strawberry-Gal3 stably expressing A549 cells (A and B), HMEC-1 cells (C and D) and HUVEC cells (E) were cultured on gridded-glass bottom dishes, and then infected with GAS for 1 h. Cells were fixed and stained with DAPI for confocal microscopy. GFP-LC3 and Strawberry-Gal3 double-positive GAS were selected as targets for transmission electron microscopy. Black arrowheads indicate isolation membrane (double membrane structure), black arrows indicate multiple membrane structures inside the LC3/Gal3-decorated single membrane indicated by white arrowheads.(TIF) ppat.1006444.s003.tif (4.8M) GUID:?44411E8D-EF7B-49D0-A8CD-580F3D2B76D1 S4 Fig: LC3 and/or LAMP1-positive GAS multiplies more in endothelial cells than endothelial cells. (A) The defect in GAS clearance in endothelial cells is usually correlated with accumulation of LC3- and LAMP1-positive GAS. Both A549 and HMEC-1 cells were positive for LC3 and LAMP1. At 1 h post-infection with GAS, cells were fixed and immunostained with anti-LC3 and anti-LAMP1 antibodies. Scale bar, 10 m. (B) Intracellular GAS with LC3 (Top) or LAMP1 (bottom) were counted at the indicated time points post-infection. All quantitative data represent means SD from three impartial experiments; more than 100 cells were counted in each sample.(TIF) ppat.1006444.s004.tif (1.2M) GUID:?3E644DB0-4200-443F-9187-4C7A20178E28 S5 Fig: Recruitment of autophagy-related proteins to bacteria. Cells with ectopic expression of indicated GFP-tagged proteins were infected with GAS (A) or (B) for 1 h, and then examined for GFP signal on GAS within their cytoplasm. Images were acquired by confocal Pedunculoside microscopy. Scale bars, Pedunculoside 10 m. Percentages of ATG9-GFP positive were shown in (B). All quantitative data represent means SD from three impartial experiments.(TIF) ppat.1006444.s005.tif (3.4M) GUID:?A6147163-2355-43BE-ADB3-73AA1CFF60D1 S6 Fig: Generation of knockout cell line using the CRISPR-Cas9 system. (A) Isolated HeLa-Kyoto cells harbor an insertion at the indicated locus in the first exon of gene. The PAM and recognition sequence are labeled in blue and green, respectively. (E) The gene, there was only one thymine insertion at nucleotide position 282 (red). PAM sequence and recognition sequence are labeled in blue and green, respectively. (D) Autophagic flux was detected by western blotting for p62 and LC3.