Fig. 4.

PARG and TRPM2 are required for Ca²⁺ gating. a Fluo-4 assay in cells silenced (siRNA-T2) or unsilenced (siRNA-C) for the trpm2 gene after 5 mM H₂O₂ (mean ± SD; n = 3). b Differences in extracellular Ca²⁺ entry after 5 mM H₂O₂ in control and ACA-treated cells (5 μM ACA with additional pretreatment of ACA for 5 min) after 1,700 s (mean ± SD; n ≥ 4; *p < 0.001; t test). c MEFs were silenced for the parg gene (siRNA-G) and subjected to Fluo-4 assay. Cells treated with a control RNA sequence (siRNA-C) were analyzed in parallel. Shown are the mean ± SD (n ≥ 3). d Western-blot analysis of AIF translocation from mitochondrial to the nuclear fraction. Control (siRNA-C) and parg silenced cells (siRNA-G) treated with 5 mM H₂O₂ or solvent controls were subjected to subcellular fractionation at 6 h. Topo-1 and MnSOD served as loading and fraction purity controls. e Control MEFs (siRNA-C) and parg-silenced cells (siRNA-G) were challenged with 5 mM H₂O₂. After 20 h, survival rates were determined by Alamar blue dye. Shown are the results as percent of the untreated control cells (mean ± SD; *p < 0.005; n = 3; t test). f MEFs were loaded with ADP-ribose (2.5 mM ADP-ribose in assay buffer + transfectant) before subjected to the Fluo-4 assay (mean ± SD; n = 3). ADP-ribose (2.5 mM) without transfectant was used as a control (mean ± SD; n = 3). g Control (siRNA-C) and trpm2-silenced cells (siRNA-T2) were loaded with ADP-ribose (2.5 mM ADP-ribose in assay buffer + transfectant) before subjected to the Fluo-4 assay (mean ± SD; n = 2). h Western-blot analysis of AIF translocation from mitochondrial to the nuclear fraction. Control (siRNA-C) and trpm2-silenced cells (siRNA-T2) treated with 5 mM H₂O₂ or solvent controls were subjected to subcellular fractionation at 6 h. Topo-1 and MnSOD served as loading controls