6.

(A) Effect of AMPK downregulation on the extent of guanosine nucleoside analogue-induced death in shRNA-transformed HSVTK⁺ BHK cells at (i) 24 hrs, (ii) 48 hrs and (iii) 72 hrs of treatment. Cell death was measured by propidium iodide staining and flow cytometry analysis. ‘Dead’ cells were counted as propidium iodide-positive events scoring above the 102 value on the FL2 scale. Data are averaged from n = 3 clones from the respective shRNA-expressing groups (green = pSilencer/negative control shRNA, blue = pSilencer/AMPKα₁shRNA, red = pSilencer/ AMPKα₂shRNA-transformed cells). Asterisk indicates significant difference between levels of cell death in the negative control shRNA and AMPKα shRNAexpressing cells, treated with 10 μM PCV or GCV (p < 0.05, Student's t-test). (B) Flow cytometry analysis of propidium iodide-stained pSilencer/negative control (i), AMPKα₁ (ii) or AMPKα₂ (iii) shRNA-transformed PC12 cells, cultured for 4 days in medium with (green plots) or without (red plots) glucose. Enhanced cell death is evident in the pSilencer/AMPKαshRNAtransformed cells. (C) Flow cytometry analysis of propidium iodide-stained, non-transformed PC12 cells, cultured for 4 days without compound C (i), with 0.1 μM compound C (ii) or with 1μM compound C (iii), in medium with (green plots) or without (red plots) glucose. Glucose deprivation-induced death is enhanced in the compound C-treated cells.