Figure 5

Confocal microscopy of pERK, MnSOD and diaphorase histochemistry on Caco-2 treated with MnGluc or MnOxP. Single plane cross-sectional images of a z-series stack from Caco-2 cells doubly labeled for MnSOD (green) and pERK (red) are in panel A left. Cross sections show x-y (squared), x-z and y-z (both rectangular) projections of the same field. Bright granular fluorescence for MnSOD with both metal supplements is shown, but only faint granules of pERK are visible, indicative of mitochondria metabolically active and of healthy cells, respectively. In panel A right the 2D free projection max obtained from all z-planes of the stack are shown, to evidence the contribution of whole cells to pERK signal and to underline the scarce total expression of pERK. βNADPH- and NADH diaphorase assays (representative experiment in panel B) demonstrates by the presence of stable blue formazan precipitate a prevalent activation of NADH- respect to βNADPH-related enzymes upon incubation with MnGluc or MnOxP, a slightly different time-course with the two compounds and a moderate but non-homogenous activation of NADH diaphorase in treated compared to untreated cells.