Figure 1.
rTSST-1 suppresses autophagy in nutrient-starved HeLa 229 cells.
HeLa 229 cells were transfected with pEGFP-hLC3 or pEGFP-C2 (Mock). Effect of TSST-1 was observed in nutrient-rich (MEM) and nutrient-starvation (KRB) condition by addition of 10 µg/ml rTSST-1. At 6 h, GFP-LC3 puncta were assessed under confocal microscope (A). GFP-LC3 puncta were counted from 100 cells of 3 independent-experiments (B).
Figure 2.
rTSST-1 does not enhance autophagosome and lysosome fusion.
HeLa 229 cells were transfected with pEGFP-hLC3. Autophagy was induced under nutrient-starvation condition for 0 and 6 h with or without the addition of 10 µg/ml rTSST-1. Lysosomes were immunostained with LAMP1, lysosomes and GFP-LC3 puncta were observed under confocal microscope (A). GFP-LC3 puncta and overlapping between GFP-LC3 and lysosomes were counted from 100 cells of 2 independent-experiments (B).
Figure 3.
Lysosomal protease inhibitors fail to restore autophagosomes in TSST-1-treated cells.
HeLa 229 cells were transfected with pEGFP/hLC3 and autophagy was induced under nutrient-starvation with or without the addition of 10 µg/ml rTSST-1 and lysosomal protease inhibitors. At the indicating time, the autophagosomal accumulation in the cells was observed by GFP puncta under confocal microscope (A). GFP-LC3 puncta at 4 h were counted from 100 cells of 2 independent-experiments (B).
Figure 4.
Suppression of autophagy by rTSST-1 is observed by immunostaining and electron microscopy.
Autophagy in HeLa 229 cells was observed in nutrient-rich (MEM) or nutrient-starvation (KRB) condition containing lysosomal protease inhibitors with or without the addition of 10 µg/ml rTSST-1. At 4 h, the cells were fixed and washed. (A) Autophagosomes were stained with anti-LC3 antibody and rhodamine-conjugated anti-rabbit IgG, and then observed under confocal microscope. (B) LC3 puncta were counted from 100 cells of 3 independent-experiments. (C) Autophagosomes were observed under electron microscope. AP indicates autophagosome-like vacuole.
Figure 5.
rTSST-1 suppresses LC3-II accumulation in the autophagy induced HeLa 229 cells.
Autophagy in HeLa 229 cells was induced by nutrient-starvation (KRB) (A) or rapamycin (B and C) with or without the addition of lysosomal protease inhibitors and rTSST-1. Cells in MEM or DMSO were used as controls. At 4 h of induction, LC3-II was detected by Western blotting (A and B). (C) The intensity of LC3-II band was quantified by normalizing with the intensity of β-tubulin band. The amount of LC3-II was calculated relatively to that from autophagic induction condition with lysosomal protease inhibitors, which set to 1.
Figure 6.
TSST-1-producing S. aureus suppresses autophagy.
HeLa 229 cells were transfected with pEGFP-hLC3 plasmid and infected with S. aureus 834 or Δtst. At 6 h of infection, S. aureus cells were immunostained as described in the Experimental procedure. GFP-LC3 and S. aureus cells were observed under confocal microscope (A). LC3-colocalized S. aureus spots are represented and indicated by upper inset and yellow arrowheads, respectively. LC3-free S. aureus spots are represented and indicated by lower inset and white arrowheads, respectively. GFP-LC3 puncta (B), S. aureus cells (C) and colocalization of S. aureus with GFP-LC3 (D) were analyzed from at least 100 cells of 3 independent-experiments.
Figure 7.
Suppression of LC3-II accumulation by rTSST-1 in the cells is superantigenic activity-independent.
Autophagy in HeLa 229 cells was induced by nutrient-starvation (KRB) with or without lysosomal protease inhibitors and 10 µg/ml rTSST-1, mTSST-1, SEA, SEB or SEC. Cells in MEM were used control. At 4 h of induction, LC3-II was detected by Western blotting (A) and the intensity of LC3-II band was quantified (B) as described in Figure 5C. The data is provided as SD of at least 3-independent experiments.