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. 2009 Oct 26;285(3):2120–2129. doi: 10.1074/jbc.M109.065813

FIGURE 4.

FIGURE 4.

Induction of autophagy is required for effective trafficking of LeTx. A, LeTx induced the formation of intracellular vacuoles in macrophages. RAW264.7 macrophages were incubated in absence or presence of LeTx (250 ng/ml LF and 500 ng/ml PA) at 37 °C for 1 h and washed twice with ice-cold PBS. Cells were then fixed with 2.5% glutaraldehyde in 0.1 m sodium cacodylate buffer, and grids with specimen were prepared as described under “Experimental Procedures.” Micrographs were taken with a transmission electron microscope. Scale bars show 2 μm in length for the lower magnification and 0.2 μm for higher magnification. B, LeTx induced LC3-II formation in a PA-dependent manner. RAW264.7 macrophages were treated with PA and LF (PA + LF, 500 ng/ml PA and 250 ng/ml LF), PA and inactive LF (PA + mLF, 500 ng/ml PA and 250 ng/ml LF), PA only (500 ng/ml PA), or LF only (250 ng/ml PA) for 2 h. Induction of autophagy was analyzed by immunoblotting against LC3-II. An immunoblot for p38 MAPK was used as a loading control. C, RAW264.7 cells were pretreated with CA074-Me (50 or 100 μm) or the microtubule inhibitor vinblastine (0.25 μm) and treated with LeTx (125 ng/ml LF and 250 ng/ml PA) for 2 h. LC3-II accumulation was analyzed by Western blots. D, N-terminal GFP-conjugated LC3 was transiently transfected in RAW264.7 macrophages and treated with LeTx (250 ng/ml LF and 500 ng/ml PA) for 1 h at 16 h post-transfection. Cell were washed twice with normal media and further incubated at 37°C for 30 min. Cells were then fixed and immunostained with anti-PA or anti-LF as described under “Experimental Procedures.” GFP-conjugated LC3 and PA (top panel) or LF (bottom panel) were visualized using a Zeiss LSM510 META confocal microscope; scale bar, 5 μm. E, THP-1 cells were incubated with serum or without serum for 4 h and treated with LeTx (125 ng/ml LF and 250 ng/ml PA) for the indicated times. MEK1 degradation or LC3-II formation was analyzed by Western blots. Immunoblot for actin was used as a loading control. NT, N terminus. F, THP-1 cells were treated with different doses of 3-MA for 1 h and exposed by LeTx for 2 h. PA-heptamer and MEK1 degradation, phospho-ERK (pERK), and LC3-II formation were analyzed by Western blots.