Figure 1.

Mouse RDH11 and RDH12 protect HEK-293 cells against 4-HNE-induced apoptosis. Cells were transfected with the expression plasmid pTarget, containing or not Flag-tagged RDH11 or RDH12. Stable cell lines were established and maintained by growing the cells in complete DMEM medium containing G418 (1mg/ml). A, Equal aliquots (50 μg) of cell homogenates were analyzed by Western-blot using anti-Flag antibody (1:1000 dilution) to compare expression levels of RDH11 and RDH12. B, Cells were treated over night (20 h) with indicated concentrations of 4-HNE in complete DMEM containing G418. Pictures of cell dishes subjected to 0 or 100 μM of 4-HNE were taken the next day, after removing the media containing floating cells, and replacing it with PBS. Note complete absence of adhering cells in the mock-transfected clone, in contrast with RDH11- and RDH12-transfected clones in which a significant number of cells are still attached to the dish. C, Quantification of Annexin V positive cells corresponding to apoptotic cells, by flow cytometry. After 20 h treatment with 4-HNE, floating and attached cells are pooled, washed in PBS, and stained with Annexin V-PE before analysis by flow cytometry. Graph shows results of 6 independent experiments. Data points represent the mean and error bars denote SEM. RDH-expressing clones were compared with the control using the Student’s t test for significance. *=p<0.05; **=p<0.001; and ***=p<0.0001.