Microscopic images. We sought to verify the MTT results by collecting microscopic images of cells treated with 60 mM of the conjugates. The images correlated well with the cell viability data. Images of (RX) 8 À, (rX) 8 À, (RXR) 3 RBRÀ (4c), (RXR) 4 ÀPMO and vehicle-treated cells are shown in Figure 7 . Cells treated with (RX) 8 ÀPMO and (RXR) 4 ÀPMO appeared rounded and detached from the culture well, and appeared to have fewer live cells. Interestingly, cells treated with (rX) 8 ÀPMO appeared to have normal morphology and cell density. The replacement of one X of (RXR) 4 ÀPMO . Nuclear activity of CPP-PMO conjugates: number and position of X residues. pluc705 cells were treated with the conjugates having 0, 2, 3 (3a, 3b, 3c and 3d), 4 (4a, 4b and 4c), 5 and 8 X residues (see sequences in Table 1 ) in OptiMEM medium with 10% serum for 24 h. Nuclear activity of a conjugate is indicated by relative luciferase activity (RLU) per microgram of protein. Data represent a mean AE SD of 9-12 data points from four independent experiments. Propidium iodine exclusion assay. The effect of the conjugates on integrity of cell membranes was investigated by a propidium iodine (PI) exclusion assay. PI can only permeate unhealthy/damaged membranes, so positive PI fluorescence indicates compromised cell membranes. Only (RXR) 4 À and (RX) 8 ÀPMO conjugates were found to significantly affect membrane integrity at higher concentrations (up to 60 mM tested). Figure 8A shows the histograms of pLuc705 cells treated with (RXR) 4 ÀPMO at 60 mM for 0.5, 5 and 24 h. The PI positive (PI+) region was defined by the cells permeabilized with ethanol (positive control) as indicated by the gate in the histogram. The PI histogram shifts from the PI-negative region to PI-positive region in the longer incubations, indicating the conjugate caused membrane leakage in a time-dependent manner. The 0.5-and 5-h-treatments caused a slight shift towards the PI+ region, while the 24-htreatment produced a distinct peak which corresponded to 57% of cells that were in the PI+ region. Figure 8B shows the histograms of cells treated with (RXR) 4 ÀPMO at concentrations of 2, 10, 20, 40 and 60 mM for 24 h. There was no significant PI uptake at concentrations up to 20 mM. At higher concentrations, the PI+ population appeared and the percentage of PI+ cells increased as the treatment concentration increased, indicating that there were more leaking cells at the higher treatment concentration. Similar concentrationand time-dependent PI uptake profiles were observed for (RX) 8 ÀPMO but not for (RB) 8 ÀPMO and the remaining conjugates (data not shown). Addition of 10% serum to the treatment medium significantly reduced membrane toxicity for the (RXR) 4 À ( Figure 8C ) and (RX) 8 ÀPMO conjugates (data not shown).