Figure 3

Detection of apoptosis induction in U-2 OS cells seeded on an adenoviral microarray. A. Relative expression levels of the target genes indicated after co-transfection of the psiCHECK marker plasmid and shRNA-encoding plasmids directed against the target genes indicated. B. TUNEL assay (left part of the image) or immunofluorescence of anti-cleaved caspase 3 (right part of the figure) of cells expressing EGFP and either control shRNA (left) or shRNA directed against p38alpha (right). Top, green fluorescence indicating infected cells; centre, red fluorescence for detection of TUNEL-positive cells or cells positive for cleaved caspase 3; bottom, merged images. Scale bar, 20 μm (for TUNEL) and 10 μm (for cleaved caspase 3 immunostaining). C. Functional assays performed on the microarray, using shRNA sequences directed against the target genes indicated. Caspase 3 (black bars) or TUNEL signal (hatched bars) were quantified in U-2 OS cells (ImageJ software). The statistical significance is marked by asterisks (4 spots, n > 150, p < 0.005, three independent experiments; by ANOVA analysis). D. Comparison of caspase 3 activation using either immunodetection on the microarray (black bars), or detection of enzymatic caspase 3 activity in cell lysates (Caspase-3-Glo, in a 96-well format (hatched bars). Statistical significance is marked by asterisks (4 spots, n > 150, p < 0.005, three independent experiments; by ANOVA analysis). On the right, the calculations of signal-to-noise ratios are shown.