Figure 6

Antiapoptosis by acridine orange staining of the UB09/RON cells after MTX treatment. (A) UB09 cells were electroporated with 40 μg of pLP-TRE2-RON and 40 μg of pTet-Lac-Hyg for 12 h. Cells were first cultured in serum-containing medium for 12 h. Then that medium was replaced with medium containing only MTX (50 μg ml⁻¹), MTX and doxycycline (1 μg ml⁻¹), and, finally, wortmannin (10 nM), MTX, and doxycycline for 60 h each. All the cells were fixed with methanol for 15 min and stained with 1% acridine orange for 10 min. Cells were examined using fluorescent microscopy (left: × 100; right: × 200). Apoptotic cells are indicated by arrows. (B) Levels of apoptotic cells measured using apoptotic body calculation in three different fields were compared in relation to doxycycline or wortmannin treatment (P=0.024, t-test).