Figure 4
MTX suppresses autophagosome formation in SCC-15, but fails to inhibit autophagy in SCC-9 cells. (A) SCC-15 and SCC-9 cells were transiently transfected with GFP-tagged LC3-expressing construct or a control GFP vector by electroporation. After culturing for 18 h, cells were treated with or without 10 μg/ml MTX. Twenty-four hours later, cells were fixed with 3.7% formalin/PBS at 4 °C for 15 min and examined for punctated GFP-LC3 protein by confocal fluorescence microscopy. Scale bar, 20 μm. (B) SCC-15 and SCC-9 cells were treated with or without 10 μg/ml MTX for 24 h. The presence of autophagic vacuoles in these cells was monitored by transmission electron microscopy. The enlarged images at the right panel (a–g) are from the boxed areas in the left panel. Disappearance of autophagosomes (arrows) post MTX treatment was examined in SCC-15 cells. In comparison with the normal mitochondrial ultrastructure (open arrowheads) in untreated SCC-15 cells, numerous intensely stained mitochondria with swollen cristae (solid arrowheads) were observed in MTX-treated SCC-15 cells. In contrast, the autophagosome number and mitochondrial morphology remained largely unchanged after MTX treatment in SCC-9 cells. Original magnification: left panel images, × 10 000 or × 5000. Scale bars, 1 μm. Cyto, cytoplasm; Neu, nucleus
