Knockout of Beclin 1 inhibits GAS internalization.

<p>(A) Invasion rate of GAS in Beclin 1 KO cells. Both wild-type and Beclin 1 KO cells were infected with GAS (MOI = 100). At 1 h post-infection, cells were disrupted with distilled water and serial dilutions of cellular extracts were plated on THY agar plates, and colony counting was performed. The data presents the invasion rate as the ratio of “total intracellular GAS at 2 h post-infection” to “total adherent GAS at 1 h post-infection”. Data are representative of ≥ three independent experiments. ** <i>P</i> < 0.01. (B) The number of cells containing Galectin-3-positive GAS were counted and presented as the percentage of the total number of GAS-infected cells. Wild-type and Beclin 1 KO cells stably expressing GFP-LC3 were infected with GAS (MOI = 100) for the indicated times. Cells were then immunostained with an anti-Galectin-3 antibody. Cellular and bacterial DNA was stained with DAPI. The data shown represent results from >200 infected cells in terms of the mean value ± SD from three independent experiments. * <i>P</i> < 0.05, ** <i>P</i> < 0.01. (C) Confocal microscopic images of Galectin-3-positive GAS at 4 h after infection. White arrow heads show Galectin-3-positive GAS. Scale bars, 10 μm. (D) Co-localization efficiencies of Galectin-3-positive GAS and GcAV were calculated as the percentage of total number of Galectin-3-positive GAS. Wild-type and Beclin 1 KO cells stably expressing GFP-LC3 were infected with GAS (MOI = 100) for 4 h. Cells were then immunostained with an anti-Galectin-3 antibody. Cellular and bacterial DNA was stained with DAPI. The data shown represent results of >50 Galectin-3-positive GAS and each percentage represents the mean value ± SD from three independent experiments. (E) Confocal microscopic images of LC3-positive GAS in Galectin-3-positive GAS in Beclin 1 KO cells. Insets show expansion of the boxed areas. Scale bars, 10 μm. (F) The number of cells containing GcAV were counted and presented as the percentage of the total number of GAS-infected cells. Wild-type and Beclin 1 KO cells stably expressing GFP-LC3 were infected with GAS (MOI = 100) for 4 h. Cellular and bacterial DNA was stained with DAPI. The data shown represent results from >200 infected cells in terms of the mean value ± SD from three independent experiments. (G) Confocal microscopic images of GcAV in Beclin 1 KO cells. White arrowheads show GcAVs. Scale bars, 10 μm. (H) The accumulation of LC3-II in Beclin 1 KO cells. Wild-type and Beclin 1 KO cells were infected with GAS (MOI = 100) for 4 h. Expression of LC3 was analyzed by western blotting using anti-LC3 antibody. (I) Co-localization efficiencies of GcAV and lysosomes in Beclin 1 KO cells. Wild-type and Beclin 1 KO cells stably expressing GFP-LC3 were infected with GAS (MOI = 100) for 4 h. Cells were then immunostained with an anti-LAMP1 antibody. Cellular and bacterial DNA was stained with DAPI. Co-localization efficiencies of GcAV and LAMP1 were calculated as the percentage of total number of GcAV. The data shown represent results of >50 GcAVs and each percentage represents the mean value ± SD from three independent experiments. (J) Confocal microscopic images of GcAV associated with lysosomes in Beclin 1 KO cells. Insets show expansion of the boxed areas. Scale bars, 10 μm. (K) Survival rate of GAS in Beclin 1 KO cells. Both wild-type and Beclin 1 KO cells were infected and treated as in (A). The data presents the survival rate as the ratio of “intracellular live GAS at 4 h post-infection” to “total intracellular GAS at 2 h post-infection”. Data are representative of ≥ three independent experiments.</p

Bcl-xL suppresses GAS internalization.

<p>(A) The number of cells containing GcAV were counted and presented as the percentage of the total number of GAS-infected cells. HeLa cells stably expressing GFP-LC3 were transfected with FLAG-control, -Bcl-2, or -Bcl-xL and infected with GAS (MOI = 100) for 4 h. Cellular and bacterial DNA was stained with DAPI. The data shown represents result from >200 infected cells in terms of the mean value ± SD from three independent experiments. * <i>P</i> < 0.05. (B) Confocal microscopic images of GcAV in cells expressing either FLAG-control, -Bcl-2, or–Bcl-xL. Insets show expansion of the boxed areas. Scale bars, 10 μm. (C) The accumulation of LC3-II during GAS infection. HeLa cells were transfected with FLAG-control, -Bcl-2, or -Bcl-xL and infected with GAS (MOI = 100) for 4 h. Expression of LC3 was analyzed by western blotting using anti-LC3 antibody. (D) Co-localization efficiencies of GcAV and lysosomes were calculated as the percentage of the total number of GcAV. HeLa cells stably expressing GFP-LC3 were transfected with FLAG-control, or -Bcl-xL and infected with GAS (MOI = 100) for 4 h. Cells were then immunostained with an anti-LAMP1 antibody. Cellular and bacterial DNA was stained with DAPI. The data shown represent results of >50 GcAVs and each percentage represents the mean value ±SD from three independent experiments. (E) Confocal microscopic images of GcAV associated with lysosomes. White arrows show autophagosomes, white arrow heads show autolysosomes. Scale bars, 10 μm. (F) Intracellular survival rate of GAS. HeLa cells were transfected with FLAG-control, or -Bcl-xL, and infected with GAS (MOI = 100). After 1 h of infection, cells were washed with PBS and further incubated for 1 h with DMEM/10%
FCS with gentamicin (100 μg/ml) to kill the extracellular bacteria. Cells were disrupted with distilled water and serial dilutions of cellular extracts were plated on THY agar plates, and colony counting was performed. The data presents the survival rate as the ratio of “intracellular live GAS at 4 h post-infection” to “total intracellular GAS at 2 h post-infection”. Data are representative of ≥ three independent experiments. (G) Invasion rate of GAS. HeLa Cells were transfected and infected as in (F). The data presents the invasion rate as the ratio of “total intracellular GAS at 2 h post-infection” to “total adherent GAS at 1 h post-infection”. Data are representative of ≥ three independent experiments. * <i>P</i> < 0.05. (H) The number of cells containing Galectin-3-positive GAS were counted and presented as the percentage of the total number of GAS-infected cells. HeLa cells stably expressing GFP-LC3 were transfected with either FLAG-control, -Bcl-2, or–Bcl-xL and infected with GAS (MOI = 100) for 4 h. Cells were then immunostained with an anti-Galectin-3 antibody. Cellular and bacterial DNA was stained with DAPI. The data shown represent results from >200 infected cells in terms of the mean value ± SD from three independent experiments. * <i>P</i> < 0.05. (I) Confocal microscopic images of Galectin-3-positive GAS. White arrow heads show Galectin-3-positive GAS. Scale bars, 10 μm. (J) Co-localization efficiencies of Galectin-3-positive GAS and GcAV were calculated as the percentage of the total number of Galectin-3-positive GAS. HeLa cells stably expressing GFP-LC3 were transfected with either FLAG-control, -Bcl-2, or–Bcl-xL, and infected with GAS (MOI = 100) for 4 h. Cells were then immunostained with an anti-Galectin-3 antibody. Cellular and bacterial DNA was stained with DAPI. The data shown represent results of >50 Galectin-3-positive GAS and each percentage represents the mean value ± SD from three independent experiments. * <i>P</i> < 0.05. (K) Confocal microscopic images of LC3-positive GAS in Galectin-3-positive GAS. Insets show expansion of the boxed areas. Scale bars, 10 μm.</p

Muistitietotutkimusmenetelmän käyttö hoitotieteellisissä tutkimuksissa : esimerkkinä lääkintälottien kokemukset koulutuksesta ja sota-ajan sairaanhoidosta

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