Fig. 3.

Effects of H₂O₂ on cytosolic Ca²⁺ and Ca²⁺ signaling. a Microscopic visualization of intracellular Ca²⁺ shifts after 5 mM H₂O₂ (3 h) using the Ca²⁺ indicator Fluo-4 in wild-type and parp-1 ^−/− cells. b Western-blot analysis of phosphorylated PERK protein with α-tubulin as loading control. Wild-type (wt P1) and parp-1 knockout MEFs (P1^−/−) were treated for the indicated timepoints with 5 mM H₂O₂. c Western-blot analysis of phosphorylated CaMKII in H₂O₂ (5 mM) treated wild-type (wt P1) and parp-1 ^−/− MEFs (P1^−/−) at the indicated timepoints. CaMKII is shown as a loading control. d Western-blot analysis of α-fodrin in wild-type (wt P1) and parp-1 knockout (P1^−/−) cells. Cells were treated with 5 mM H₂O₂ for the indicated time points, harvested and subjected to Western-blot analysis. GAPDH is loading control. e Activation of caspases and appearance of cleaved PARP-1 after 5 mM H₂O₂ is shown in Western-blot analysis of whole cell lysates prepared from wild-type (wt P1) and parp-1 knockout (P1^−/−) cells at the indicated timepoints. GAPDH and α-tubulin are shown as loading controls. f Translocation of AIF after H₂O₂ insult was determined in wild-type cells silenced for the parp-1 gene by RNAi (siRNA 1) and cells silenced with a scrambled control siRNA (siRNA C). After 6 h treatment with 5 mM H₂O₂, cells were subjected to subcellular fractionation and immunoblotting. Topo-1 and MnSOD protein are also shown