FIGURE 6.

Effect of CHX and long-term (24-h) tocopherol exposure of hTERT-RPE cells on tBHP-mediated cytotoxicity. hTERT-RPE cells were exposed to CHX for 1 h followed by αT or γT for 24 h prior to tBHP. (A) Image panels showing DAPI labeling of nuclei (blue) in cells exposed to CHX, αT or γT and tBHP. Top row represents conditions without CHX while the bottom row represents conditions with CHX. Nucleus area (blue fluorescence) is reduced during cell death by a process resembling apoptosis. Images acquired at 40x magnification; scale bar is 100 μm. (B) Measurement of pyknotic nuclei, nuclei that are half the size of average normal nuclei and not merely reduced in size, was carried out on CHX-, tocopherol- and tBHP-exposed cells. Exposure of cells to CHX (dark bars) alone increases the percentage of pyknotic nuclei in the cell population from 4% in control cells (light bars) to 17%. Exposure of cells to tBHP led to an increase in the percentage of pyknotic nuclei from 4 to 40%. Exposure of cells to either tocopherol alone had no effect on the percentage of pyknotic nuclei, but exposure to CHX and tocopherols led to an increase in the percentage of pyknotic nuclei from 2 to 28% for αT and 3–34% for γT. The presence of αT prior to tBHP insult reduced the percentage of pyknotic nuclei by 57% compared to cells treated with tBHP alone. Inhibition of protein synthesis by CHX partially reversed this αT protection by 47%. The presence of γT prior to tBHP insult reduced the percentage of pyknotic nuclei by 87% compared to cells treated with tBHP alone. Inhibition of protein synthesis by CHX partially reversed this γT protection by 44%. Microfluorimetric analyses are from an average of three images. **p ≤ 0.01 with CHX versus the same condition without CHX, ^## p ≤ 0.01 with tBHP versus the same condition without tBHP as determined by one-way ANOVA analysis with Bonferroni post hoc test.